RESUMO
Cytochrome P450 2D6 (CYP2D6) is a key enzyme that mediates the metabolism of various drugs and endogenous substances in humans. However, its biological role in drug-drug interactions especially mechanism-based inactivation (MBI), and various diseases remains poorly understood, owing to the lack of molecular tools suitable for selectively monitoring CYP2D6 in complex biological systems. Herein, using a tailored molecular strategy, we developed a fluorescent probe BDPM for CYP2D6. BDPM exhibits excellent specificity and imaging capability for CYP2D6, making it suitable for the real-time monitoring of endogenous CYP2D6 activity in living bio-samples. Therefore, our tailored strategy proved useful for constructing the highly selective and enzyme-activated fluorescent probes. BDPM as a molecular tool to explore the critical roles of CYP2D6 in the pathogenesis of diseases, high-throughput screening of inhibitors and intensive investigation of CYP2D6-induced MBI in natural systems.
RESUMO
Tuberculosis (TB) is a chronic systemic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). Methionine aminopeptidase 1 (MtMET-AP1) is a hydrolase that mediates the necessary post-translational N-terminal methionine excision (NME) of peptides during protein synthesis, which is necessary for bacterial proliferation and is a potential target for the treatment of tuberculosis. Based on the functional characteristics of MtMET-AP1, we developed an enzymatic activated near-infrared fluorescent probe DDAN-MT for rapid, highly selective, and real-time monitoring of endogenous MtMET-AP1 activity in M. tuberculosis. Using the probe DDAN-MT, a visually high-throughput screening technique was established, which obtained three potential inhibitors (GSK-J4 hydrochchloride, JX06, and lavendustin C) against MtMET-AP1 from a 2560 compounds library. More importantly, these inhibitors could inhibit the growth of M. tuberculosis H37Ra especially (MICs < 5 µM), with low toxicities on intestinal bacteria strains and human cells. Therefore, the visual sensing of MtMET-AP1 was successfully performed by DDAN-MT, and MtMET-AP1 inhibitors were discovered as potential antituberculosis agents.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Antituberculosos/farmacologia , Mycobacterium tuberculosis/metabolismo , Corantes Fluorescentes , Testes de Sensibilidade Microbiana , Aminopeptidases/metabolismoRESUMO
Lipase found in the gut microbiota participates in the digestion and absorption of dietary fats. As such, the gut microbiota is involved in the regulation of the host metabolism, affecting the levels of lipids and free fatty acids, ultimately resulting in obesity. In this study, an enzymatic activatable near-infrared fluorescent probe, DDAO-C6, was developed for visually sensing endogenous lipase from gut microbes. Using DDAO-C6, a cultivated intestinal yeast strain was rapidly identified from human feces that exhibited high lipase expression and was identified as Trichosporon asahii Y2. We then determined that the colonization of the gut of mice with T. asahii Y2 increased lipase activity in the digestive tract and promoted obesity and hyperlipidemia when the mice were fed high fat diets. Above all, the present research resulted in a fluorescence visualization tool for the functional investigation of gut microbiota associated with obesity and disorders of lipid metabolism.
Assuntos
Basidiomycota , Corantes Fluorescentes , Obesidade , Animais , Basidiomycota/classificação , Dieta Hiperlipídica , Humanos , Lipase , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/microbiologia , LevedurasRESUMO
CYP2J2 as an endoplasmic reticulum (ER)-expressed vital cytochrome P450 isoform participates in the metabolism of endogenous polyunsaturated fatty acids. Its abnormal expression and function are closely related to the progress of cancer and cardiovascular diseases. Herein, an ER-targeting near-infrared (NIR) fluorescent probe ER-BnXPI was developed for monitoring CYP2J2 activity, which possessed a high selectivity and sensitivity toward CYP2J2 among various CYP450 isoforms and exhibited excellent subcellular localization for ER. Then, the CYP2J2 variation behavior under the ER stress model was imaged by ER-BnXPI in living cells and successfully used for the in vivo imaging in different tumors that well distinguished tumor tissues from para-cancerous tissues. All these findings fully demonstrated that ER-BnXPI could be used as a promising tool for exploring the physiological function of CYP2J2 and provided some novel approach for the diagnosis and therapy of CYP2J2-related vascular inflammation and cancer.
Assuntos
Corantes Fluorescentes , Neoplasias , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Corantes Fluorescentes/metabolismo , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismoRESUMO
A rapid test method for the determination of pesticide toxicity was established by using carboxylesterase (CES) and fluorescence probe ACE-NH based on the principle of enzyme inhibition, and this method was applied to detect the combined toxicity of 18 binary and 24 ternary pesticide combinations commonly used for fruits and vegetables to CES. The results show that chlorpyrifos + carbendazim, carbofuran + carbendazim, imidacloprid + carbendazim, imidacloprid + dimethomorph, dimethoate + dimethomorph, prochloraz + carbendazim and imidacloprid + acetamiprid + carbendazim had synergistic effects under three concentration gradients, it indicated that most binary combinations containing carbendazim or imidacloprid had synergistic effects. Based on structure-activity relationship between pesticides and CES, pesticides with phosphate ester bonds had great toxicity to CES, or though they have no toxicity to CES alone, they showed a strong synergistic effect when mixed with other pesticides. Pesticides with amide or ester bond had medium toxicity and little synergistic effect. Pesticides with urea, carbamate or nitrite nitrogen group had little or no toxicity, while there was a strong synergistic effect after mixing with other pesticides. The test method and results in this study can provide scientific basis for risk assessment of cumulative exposure to mixed pesticide residues.
Assuntos
Praguicidas , Carboxilesterase , Ésteres , Fluorescência , Corantes Fluorescentes , Praguicidas/toxicidade , TecnologiaRESUMO
Mechanism-based inactivation (MBI) can mediate adverse reactions and hepatotoxicity from drugs, which is a result of their conversion into highly reactive metabolites catalyzed by enzymes such as cytochrome P450 3A (CYP3A). In the present research, we optimized the key interaction domain of the fluorophore with the target protein to develop a two-photon fluorescent probe for CYP3A that is involved in the metabolism of more than half of all clinical drugs. The developed BN-1 probe exhibited appropriate selectivity and sensitivity for the semi-quantitative detection and imaging of endogenous CYP3A activity in various living systems, thereby providing a high-throughput screening system enabling evaluation of MBI-associated hepatotoxicity by CYP3A. Using BN-1 as a fluorescent molecular tool facilitates the efficient discovery and characterization of CYP3A-induced MBI in natural systems.
Assuntos
Citocromo P-450 CYP3ARESUMO
UDP-glucuronosyltransferase 1A1 (UGT1A1) is a vital metabolic enzyme responsible for the clearance of endogenous substances and drugs. Hitherto, the development of fluorescent probes for UGTs was severely restricted due to the poor isoform selectivity and on-off or blue-shifted fluorescence response. Herein, we established a novel "molecular-splicing" strategy to construct a highly selective near-infrared (NIR) fluorescent probe, HHC, for UGT1A1, which exhibited a NIR signal at 720â nm after UGT1A1 metabolism. HHC was then successfully used for the real-time imaging of endogenous UGT1A1 in living cells and animals and to monitor the bile excretion function. In summary, an isoform-specific NIR fluorescent probe has been developed for monitoring UGT1A1 activity in living systems, high-throughput screening of novel UGT1A1 inhibitors and visual evaluation of bile excretion function.
Assuntos
Corantes Fluorescentes/química , Glucuronosiltransferase/metabolismo , Animais , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Corantes Fluorescentes/metabolismo , Vesícula Biliar/metabolismo , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/genética , Células Hep G2 , Humanos , Fígado/metabolismo , Camundongos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Sophora/química , Sophora/metabolismo , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Fatty acid amide hydrolase (FAAH) is an important drug target for the treatment of many disease related conditions such as pain, inflammation, and mood disorders due to its vital role in the metabolism of endocannabinoid. In our present work, a FAAH-activated fluorescent probe named THPO was developed, which possessed high selectivity and excellent sensitivity for FAAH in complex systems. Critically, its metabolite 7-amino-3H-phenoxazin-3-one (AHPO) has long excitation and emission wavelengths and high fluorescence quantum yield, which are necessary for monitoring the activity of FAAH in living systems. In addition, a visual high-throughput screening method for FAAH inhibitors was established using THPO, which resulted in the discovery of an efficient natural inhibitor Neobavaisoflavone that was identified from 68 traditional herbal medicines. These results indicated that THPO can be used as a molecular tool for the rapid evaluation of FAAH activity in complex systems as well as providing an effective approach to screen FAAH inhibitors and providing a boost for the discovery of therapeutic agents toward FAAH related diseases.
Assuntos
Amidoidrolases/antagonistas & inibidores , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/métodos , Isoxazóis/química , Isoxazóis/farmacologia , Benzamidas/farmacologia , Carbamatos/farmacologia , Linhagem Celular , Sobrevivência Celular , Humanos , Isoxazóis/metabolismo , Psoralea/química , Proteínas RecombinantesRESUMO
Cytochrome P450 2J2 (CYP2J2), a key enzyme responsible for oxidative metabolism of various xenobiotics and endogenous compounds, participates in a diverse array of physiological and pathological processes in humans. Its biological role in tumorigenesis and cancer diagnosis remains poorly understood, owing to the lack of molecular tools suitable for real-time monitoring CYP2J2 in complex biological systems. Using molecular design principles, we were able to modify the distance between the catalytic unit and metabolic recognition moiety, allowing us to develop a CYP2J2 selective fluorescent probe using a near-infrared fluorophore ( E)-2-(2-(6-hydroxy-2, 3-dihydro-1 H-xanthen-4-yl)vinyl)-3,3-dimethyl-1-propyl-3 H-indol-1-ium iodide (HXPI). To improve the reactivity and isoform specificity, a self-immolative linker was introduced to the HXPI derivatives in order to better fit the narrow substrate channel of CYP2J2, the modification effectively shortened the spatial distance between the metabolic moiety ( O-alkyl group) and catalytic center of CYP2J2. After screening a panel of O-alkylated HXPI derivatives, BnXPI displayed the best combination of specificity, sensitivity and applicability for detecting CYP2J2 in vitro and in vivo. Upon O-demethylation by CYP2J2, a self-immolative reaction occurred spontaneously via 1,6-elimination of p-hydroxybenzyl resulting in the release of HXPI. Allowing BnXPI to be successfully used to monitor CYP2J2 activity in real-time for various living systems including cells, tumor tissues, and tumor-bearing animals. In summary, our practical strategy could help the development of a highly specific and broadly applicable tool for monitoring CYP2J2, which offers great promise for exploring the biological functions of CYP2J2 in tumorigenesis.
Assuntos
Sistema Enzimático do Citocromo P-450/análise , Corantes Fluorescentes/química , Xantenos/química , Animais , Linhagem Celular Tumoral , Citocromo P-450 CYP2J2 , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Leucemia/diagnóstico por imagem , Linfoma/diagnóstico por imagem , Camundongos Nus , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Neovascularização Patológica/diagnóstico por imagem , Ligação Proteica , Xantenos/síntese química , Xantenos/metabolismoRESUMO
Rational design of practical probes with excellent specificity and improved optical properties for a particular enzyme is always a big challenge. Herein, a practical and highly specific fluorescent probe for carboxylesterase 1 (CES1) was rationally designed using meso-carboxyl-BODIPY as the basic fluorophore based on the substrate preference and catalytic properties of CES1. Following molecular docking-based virtual screening combined with reaction phenotyping-based experimental screening, we found that MMB (probe 7) exhibited the optimal combination of sensitivity and specificity toward human CES1 in contrast to other ester derivatives. Under physiological conditions, MMB could be readily hydrolyzed by CES1 and release MCB; such biotransformation brought great changes in the electronic properties at the meso position of the fluorophore and triggered a dramatic increase in fluorescence emission around 595 nm. Moreover, MMB was cell membrane permeable and was successfully applied to monitor the real activities of CES1 in various biological samples including living cells, tissue slices, organs, and zebrafish. In summary, this study showed a good example for constructing specific fluorescent probe(s) for a target enzyme and also provided a practical and sensitive tool for real-time sensing of CES1 activities in complicated biological samples. All these findings would strongly facilitate high-throughput screening of CES1 modulators and the studies on CES1-associated physiological and pathological processes.
Assuntos
Carboxilesterase/metabolismo , Desenho de Fármacos , Corantes Fluorescentes/química , Animais , Compostos de Boro/química , Células CACO-2 , Carboxilesterase/química , Humanos , Hidrólise , Modelos Moleculares , Imagem Molecular , Conformação Proteica , Peixe-ZebraRESUMO
As is well-known, fungi are an important biocatalysis model of glucosylation and have been widely applied for bioactive compounds glucosylation mediated by the intracellular glucosytransferases (GTs). However, there is no efficient method for the real-time detection of GTs and the rapid isolation of the target fungi strains with the high expression of GTs. In the present work, we first developed a two-photon ratiometric fluorescent probe N-( n-butyl)-4-hydroxy-1,8-naphthalimide (NHN) for detecting the glucosyltransferases activity and intracellular imaging of GTs. Under UV light (365 nm), the transformed product of NHN mediated by intracellular glucosyltransferase displayed blue emission to guide the rapid isolation of fungal strains possessing overexpression of GTs from complex soil samples. Finally, by using the fluorescent probe, two target fungi were isolated and identified to be Rhizopus oryzae and Mucor circinelloides by molecular analysis, and they exhibited a robust capability for regio- and stereospecific O-glycosylation. Our results fully demonstrated that NHN may be a promising tool for guiding real-time GTs activity in fungal strains and even for developing natural fungal strains with GTs overexpression.
Assuntos
Corantes Fluorescentes/química , Glucosiltransferases/análise , Naftalimidas/química , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/efeitos da radiação , Glicosilação , Raios Infravermelhos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Mucor/enzimologia , Mucor/isolamento & purificação , Naftalimidas/síntese química , Naftalimidas/efeitos da radiação , Rhizopus/enzimologia , Rhizopus/isolamento & purificaçãoRESUMO
Bacterial γ-glutamyltranspeptidases (γ-GT) is a well-known metabolic enzyme, which could cleave the γ-glutamyl amide bond of γ-glutamyl analogues. As a key metabolic enzyme of bacteria and a virulence factor for the host, bacterial γ-GT was determined to be a novel pharmaceutical target for new antibiotics development. However, there is no efficient method for the sensing of γ-GT activity in bacteria and the recognition of γ-glutamyltransferase rich-bacteria. In the present work, a dicyanoisophorone derivative (ADMG) has been designed and developed to be a sensitive and selective near-infrared fluorescent probe for the sensing of bacterial γ-GT. ADMG not only sensed bacterial γ-GT in vitro, but also imaged intestinal bacteria in vivo. More interesting, the intestinal bacteria existed in the duodenum section of mouse displayed significant fluorescence emission. Under the guidance of the sensing of γ-GT using ADMG, three intestinal bacteria strains K. pneumoniae CAV1042, K. pneumoniae XJRML-1, and E. faecalis were isolated successfully, which expressed the bacterial γ-GT. Therefore, the fluorescent probe ADMG not only sensed the endogenous bacterial γ-GT and imaged the intestinal bacteria but also guided the isolation of intestinal bacteria possessing γ-GT efficiently, which suggested a novel biological tool for the rapid isolation of special bacteria from a mixed sample.
Assuntos
Bactérias/isolamento & purificação , Proteínas de Bactérias/análise , Técnicas de Tipagem Bacteriana/métodos , Corantes Fluorescentes/química , Microbioma Gastrointestinal , gama-Glutamiltransferase/análise , Animais , Cicloexanonas/síntese química , Cicloexanonas/química , Enterococcus faecalis/isolamento & purificação , Corantes Fluorescentes/síntese química , Glutamatos/síntese química , Glutamatos/química , Klebsiella pneumoniae/isolamento & purificação , Camundongos , Microscopia ConfocalRESUMO
ß-Glucuronidase (GLU) is an important biomarker for primary cancers and intestinal metabolism of drugs or endogenous substances; however, an effective optical probe for near-infrared (NIR) monitoring in vivo is still lacking. Herein, we design an enzyme-activated off-on NIR fluorescent probe, HC-glu, based on a hemicyanine keleton, which is conjugated with a d-glucuronic acid residue via a glycosidic bond, for the fluorescent quantification and trapping of endogenous GLU activity in vitro and in vivo. The newly developed NIR probe exhibited prominent features including prominent selectivity, high sensitivity, and ultrahigh imaging resolution. It has been successfully used to detect and image endogenous GLU in various hepatoma carcinoma cells, tumor tissues, and tumor-bearing mouse models, for cancer diagnosis and therapy. Moreover, it could detect the in vivo activity of GLU in the intestinal tracts of animals including mice and zebrafish, where GLU performs a vital biological function and is mainly distributed. It could also evaluate real intestinal distribution and real-time variations of GLU in development and growth, all of which are very helpful to guide rational drug use in the clinic. Our results fully demonstrated that HC-glu may serve as a promising tool for evaluating the biological function and process of GLU in living systems.
Assuntos
Corantes Fluorescentes/química , Glucuronatos/química , Glucuronidase/metabolismo , Indóis/química , Xantenos/química , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Glucuronatos/síntese química , Glucuronidase/química , Humanos , Indóis/síntese química , Mucosa Intestinal/metabolismo , Camundongos , Microscopia Confocal/métodos , Simulação de Acoplamento Molecular , Xantenos/síntese química , Peixe-ZebraRESUMO
Visualization of endogenous disease-associated enzymes is of great clinical significance, as it could allow earlier clinical diagnosis and timely intervention. Herein, we first synthesized and characterized an enzyme-activatable near-infrared fluorescent probe, GP-DM, for determining the activity of dipeptidyl peptidase IV (DPP IV), which is associated with various pathological processes, especially in diabetes and malignant tumors. GP-DM emitted significant turn-on NIR fluorescent signals simultaneously in response to DPP IV, making it favorable for accurately and dynamically monitoring DPP IV activity in vitro and in vivo. GP-DM exhibited excellent specificity and sensitivity in DPP IV imaging, as indicated by its higher catalytic activity than other human serine hydrolases and by its strong anti-interference ability to a complex biological matrix, which was fully characterized in a series of phenotyping reactions and inhibition assays. Encouraged by the advantages mentioned above, we successfully used GP-DM to evaluate endogenous DPP IV activity in various biological samples (plasma and tissue preparations) and living tumor cells and performed real-time in vivo bioimaging of DPP IV in zebrafish and tumor-bearing nude mice. All of the results reflected and highlighted the potential application value of GP-DM in the early detection of pathologies, individual tailoring of drug therapy, and image-guided tumor resection. Furthermore, our results revealed that DPP IV, a key target enzyme, is closely associated with the migration and proliferation of cancer cells and regulating the biological activity of DPP IV may be a useful approach for cancer therapy.
Assuntos
Dipeptidil Peptidase 4/análise , Corantes Fluorescentes/química , Imagem Óptica/métodos , Animais , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidase 4/metabolismo , Ensaios Enzimáticos/métodos , Células Hep G2 , Humanos , Raios Infravermelhos , Camundongos , Microscopia Confocal/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/enzimologia , Peixe-ZebraRESUMO
In this study, a novel fluorescent detection system for biological sensing of human albumin (HA) was developed on the basis of the pseudoesterase activity and substrate preference of HA. The designed near-infrared (NIR) fluorescent probe (DDAP) could be effectively hydrolyzed by HA, accompanied by significant changes in both color and fluorescence spectrum. The sensing mechanism was fully investigated by fluorescence spectroscopy, NMR, and mass spectra. DDAP exhibited excellent selectivity and sensitivity toward HA over a variety of human plasma proteins, hydrolases, and abundant biomolecules found in human body. The probe has been successfully applied to measure native HA in diluted plasma samples and the secreted HA in the hepatocyte culture supernatant. DDAP has also been used for fluorescence imaging of HA reabsorption in living renal cells, and the results show that the probe exhibits good cell permeability, low cytotoxicity and high imaging resolution. Furthermore, DDAP has been successfully used for real-time tracking the uptaking and degradation of albumin in ex vivo mouse kidney models for the first time. All these results clearly demonstrated that DDAP-based assay held great promise for real-time sensing and tracking HA in complex biological systems, which would be very useful for basic researches and clinical diagnosis of HA-associated diseases.
Assuntos
Corantes Fluorescentes/química , Imagem Óptica , Albumina Sérica Humana/análise , Corantes Fluorescentes/síntese química , Células Hep G2 , Humanos , Raios Infravermelhos , Estrutura Molecular , Albumina Sérica Humana/biossíntese , Albumina Sérica Humana/metabolismo , Espectrometria de Fluorescência , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Carboxylesterase 1 (CE1) is an important serine hydrolase in mammals, which involved in the hydrolysis of a variety of compounds (endogenous substrates like cholesterol and xenobiotic compounds like ester-contain drugs and pesticides). This study aimed to design and develop the fluorescent probe substrates for human carboxylesterase 1 (hCE1), on the basis of the structural features of hCE1 preferred substrates. Four carboxylic esters deriving from BODIPY-8-carboxylic acid were designed and synthesized. After then, reaction phenotyping assays and chemical inhibition assays were used to evaluate the selectivity of these four ester derivatives towards hCE1. Our results clearly demonstrated that the substrate specificity of these ester substrates towards hCE1 would be improved with the decrease of the alcohol group on BODIPY-8-carboxylesters, while BODIPY-8-carboxylesters with small alcohol groups including methyl (BCM) and ethyl (BCE) esters could serve as the ideal probe substrates for hCE1. Given that BCM exhibit rapid hydrolytic rate in hCE1, we further investigate the enzymatic kinetics of this fluorescent probe substrate in both human liver microsomes (HLM) and recombinant hCE1, as well as to explore its potential application in high-throughput screening of hCE1 inhibitors by using HLM as enzyme source. The results showed that the kinetic behaviors and the affinity of BCM in HLM is much closed to those in recombinant hCE1, implying that hCE1 played the key roles in BCM hydrolysis in HLM. Furthermore, the inhibition study demonstrated that BCM could be used for rapid screening and characterization of hCE1 inhibitors, by using HLM to replace recombinant hCE1 as enzyme source.
Assuntos
Compostos de Boro/química , Hidrolases de Éster Carboxílico/química , Corantes Fluorescentes , Ésteres , Humanos , Hidrólise , Cinética , Microssomos Hepáticos/enzimologia , Especificidade por SubstratoRESUMO
Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems.
Assuntos
Citocromo P-450 CYP1A1/análise , Citocromo P-450 CYP1A2/análise , Corantes Fluorescentes/química , Fótons , Animais , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/metabolismo , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Corantes Fluorescentes/síntese química , Ensaios de Triagem em Larga Escala , Humanos , Fígado/enzimologia , Microssomos Hepáticos/enzimologia , Simulação de Acoplamento Molecular , Ratos , Células Tumorais CultivadasRESUMO
The emission intensities of coumarin 545 solution exhibit a low temperature dependence, with a record-low temperature coefficient of only â¼0.025% per °C. This monomer-aggregate coupled fluorescence system can be used for ratiometric temperature measurements with high spatial and temporal resolutions; three different working modes have been demonstrated.
Assuntos
Cumarínicos/química , Corantes Fluorescentes/química , Temperatura Baixa , Espectrometria de Fluorescência , Temperatura , TermômetrosRESUMO
Monoamine oxidase A (MAO-A) is a dimeric flavoprotein that is found in the mitochondrial membrane. Currently, there is a lack of near-infrared fluorescent probes (NIR-FPs) with good specificity and high sensitivity for detecting MAO-A, making it difficult to accurately recognize and image cells in vitro and in vivo. In this study, the NIR-FP DDM-NH2 was designed and synthesized in order to detect MAO-A specifically in live biological systems. The probe comprised two functional components: dicyanoisophosphone as an NIR dye precursor and alanine as a recognition moiety. After identifying MAO-A, the probe exhibited an NIR emission peak at 770 nm with a significant Stokes shift (180 nm), 11-fold response factor, low detection limit of 99.7 nM, and considerably higher affinity toward MAO-A than that toward MAO-B, indicating high sensitivity. In addition, DDM-NH2 was effective when applied to the image-based assessment of MAO-A activity in HeLa cells, zebrafish, and tumor-bearing mice, demonstrating great potential for visualization-based research and MAO-A application in vivo.