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1.
Anal Bioanal Chem ; 411(28): 7341-7355, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31667564

RESUMO

Over two decades, the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) has organized a number of comparisons for clinically relevant small molecule organic biomarkers. The aim of the OAWG community is to be part of the coordinated international movement towards accuracy and comparability of clinical measurements that will, in turn, minimize the wastage of repeat testing and unnecessary therapy to create a sustainable healthcare industry. International and regional directives/requirements on metrological traceability of calibrators and control materials are in place. Metrology institutes worldwide maintain infrastructure for the practical realization of metrological traceability and demonstrate the equivalence of their measurement capabilities through participation in key comparisons organized under the auspices of the CCQM. These institutes provide certified reference materials, as well as other dedicated value-assignment services benefiting the in-vitro diagnostic (IVD) industry, reference (calibration) laboratories and the clinical chemistry laboratories. The roles of these services in supporting national, regional, and international activities to ensure the metrological traceability of clinical chemistry measurements are described. Graphical abstract.


Assuntos
Biomarcadores/análise , Compostos Orgânicos/análise , Bibliotecas de Moléculas Pequenas/análise , Calibragem , Testes de Química Clínica , Humanos , Técnicas In Vitro , Padrões de Referência , Reprodutibilidade dos Testes
2.
Biotechnol Appl Biochem ; 66(4): 591-596, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31050059

RESUMO

Glucose-6-phosphate dehydrogenase (G6PDH) has been used in enzyme multiplied immunoassay technique (EMIT) assays for detecting small molecule metabolites such as cholyglycine (CG). A key parameter for successful EMIT CG assay development is the inhibition rate of the G6PDH-CG conjugate, measured as the decrease in enzyme activity upon CG antibody binding. Several commonly used G6PDH cysteine mutants including A45C and K55C have been labeled with CG-maleimide derivative, but inhibition rates of are unsatisfactory. Herein, we investigated whether other mutation sites can achieve better inhibition rates. We generated eight cysteine mutants (K106C, Y155C, A201C, T258C, D306C, D375C, G426C, and D480C) of G6PDH, measured their inhibition rates, and evaluated the performance of the D306C mutant using EMIT CG assays. One of the eight mutants (D306C) displayed improved inhibition rate, whereas all others exhibited inhibition similar to or lower than that of A45C and K55C. The enhanced inhibition rate of D306C improved the EMIT CG assay calibration curve, using an Abbott c16000 automated biochemical analyzer, resulting in better repeatability, precision, and linearity than with K55C assays and a commercially available EMIT CG kit. The G6PDH mutant D306C has a higher inhibition rate in EMIT CG assays and improves assay performance.


Assuntos
Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Ácido Glicocólico/análise , Imunoensaio , Mutação , Bibliotecas de Moléculas Pequenas/análise , Cisteína/genética , Glucosefosfato Desidrogenase/química , Ácido Glicocólico/metabolismo , Humanos , Bibliotecas de Moléculas Pequenas/metabolismo
3.
Molecules ; 24(10)2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31137582

RESUMO

In order to evaluate the quality of Bufonis Venenum commercial herbs, a three-step qualitative and quantitative research study was performed. Firstly, we tried to identify small molecules and peptides in Bufonis Venenum using pre-fractionation chromatography and high-resolution mass spectrometry. The database search of the small molecules and peptides of Bufonis Venenum revealed that the dried venom consisted of free/conjugated-type bufadienolides and peptides with a mass range of 0.4-2 kDa. Secondly, we used partial least squares (PLS) multivariate statistical analysis to screen bufadienolides markers (VIP > 1.5) responsible for the anti-tumor cell activity of Bufonis Venenum, including 21 identified bufadienolides and 7 unknown compounds. It is noticeable that these bufadienolide markers could not be recognized by traditional HPLC-UV based spectrum-effect relationship analysis (correlation coefficient ranging from -0.24 to 0.40). Finally, we proposed a weight coefficient-based corrected total contents of 9 bufadienolides as a quality evaluation indicator, which had good correlation with inhibitory effects on tumor cells of commercial Bufonis Venenum. The correlation coefficient increased from 0.4 to 0.6. Thus, our pre-fractionation chromatography and mass spectrometry strategy had significant advancement over the traditional spectrum-effect relationship method for chemical marker identification. These results could be crucial and helpful in the development of a quality evaluation method that could reflect the pharmacological activity of Bufonis Venenum.


Assuntos
Antineoplásicos/farmacologia , Bufanolídeos/análise , Bufanolídeos/farmacologia , Espectrometria de Massas , Peptídeos/análise , Peptídeos/farmacologia , Sequência de Aminoácidos , Calibragem , Linhagem Celular Tumoral , Humanos , Análise dos Mínimos Quadrados , Limite de Detecção , Análise Multivariada , Peptídeos/química , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/análise
4.
Int J Mol Sci ; 20(4)2019 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-30813400

RESUMO

The initiative strategy for the development of novel anti-microbial agents usually uses the virulence factors of bacteria as a target, without affecting their growth and survival. The type III secretion system (T3SS), one of the essential virulence factors in most Gram-negative pathogenic bacteria because of its highly conserved construct, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) causes leaf blight diseases and is one of the most important pathogens on rice. To find potential anti-virulence agents against this pathogen, a number of natural compounds were screened for their effects on the T3SS of Xoo. Three of 34 compounds significantly inhibited the promoter activity of the harpin gene, hpa1, and were further checked for their impact on bacterial growth and on the hypersensitive response (HR) caused by Xoo on non-host tobacco plants. The results indicated that treatment of Xoo with CZ-1, CZ-4 and CZ-9 resulted in an obviously attenuated HR without affecting bacterial growth and survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the three inhibitors. The mRNA levels of representative genes in the hypersensitive response and pathogenicity (hrp) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.


Assuntos
Oryza/microbiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sistemas de Secreção Tipo III/metabolismo , Xanthomonas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Genes de Plantas , Oryza/efeitos dos fármacos , Oryza/genética , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/química , Tabaco/microbiologia , Xanthomonas/efeitos dos fármacos , Xanthomonas/crescimento & desenvolvimento
5.
Talanta ; 198: 350-357, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30876572

RESUMO

Ultrasensitive Surface Plasmon Resonance (SPR) detection of small molecules can be achieved using nanoparticles to both enhance the SPR signals and pre-concentrate low levels of target analytes in the sample. However, the short effective penetration depth of the SPR evanescent field, and steric hindrance of binding when immobilizing small molecules on surfaces, limits the applicability of using relatively large nanoparticles (≥100 nm) for SPR detection. To overcome the issues of steric hindrance, this paper investigates the role of the molecular linkers to tether both the antibodies to the magnetic nanoparticles, and to bind the small molecules to the surface of the SPR chips. By extending the distance of the small molecule (progesterone) away from the SPR chip surface and improving the antibody orientation on the large magnetic nanoparticles, a sensitive SPR detection for progesterone was achieved in buffer (0.013 ng mL-1). The results of the SPR assay for progesterone in milk were in good correlation with ELISA results, and could be used to verify the onset of estrus in cows.


Assuntos
Nanopartículas/química , Progesterona/análise , Bibliotecas de Moléculas Pequenas/análise , Ressonância de Plasmônio de Superfície , Ensaio de Imunoadsorção Enzimática
6.
Nat Commun ; 10(1): 1402, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926793

RESUMO

Protein-protein interactions (PPIs) governing the recognition of substrates by E3 ubiquitin ligases are critical to cellular function. There is significant therapeutic potential in the development of small molecules that modulate these interactions; however, rational design of small molecule enhancers of PPIs remains elusive. Herein, we report the prospective identification and rational design of potent small molecules that enhance the interaction between an oncogenic transcription factor, ß-Catenin, and its cognate E3 ligase, SCFß-TrCP. These enhancers potentiate the ubiquitylation of mutant ß-Catenin by ß-TrCP in vitro and induce the degradation of an engineered mutant ß-Catenin in a cellular system. Distinct from PROTACs, these drug-like small molecules insert into a naturally occurring PPI interface, with contacts optimized for both the substrate and ligase within the same small molecule entity. The prospective discovery of 'molecular glue' presented here provides a paradigm for the development of small molecule degraders targeting hard-to-drug proteins.


Assuntos
Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Especificidade por Substrato/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , beta Catenina/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo
7.
ACS Comb Sci ; 21(5): 425-435, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-30884226

RESUMO

Robotic high-throughput compound screening (HTS) and, increasingly, DNA-encoded library (DEL) screening are driving bioactive chemical matter discovery in the postgenomic era. HTS enables activity-based investigation of highly complex targets using static compound libraries. Conversely, DEL grants efficient access to novel chemical diversity, although screening is limited to affinity-based selections. Here, we describe an integrated droplet-based microfluidic circuit that directly screens solid-phase DELs for activity. An example screen of a 67 100-member library for inhibitors of the phosphodiesterase autotaxin yielded 35 high-priority structures for nanomole-scale synthesis and validation (20 active), guiding candidate selection for synthesis at scale (5/5 compounds with IC50 values of 4-10 µM). We further compared activity-based hits with those of an analogous affinity-based DEL selection. This miniaturized screening platform paves the way toward applying DELs to more complex targets (signaling pathways, cellular response) and represents a distributable approach to small molecule discovery.


Assuntos
DNA/química , Bibliotecas de Moléculas Pequenas/análise , Técnicas de Química Combinatória , Avaliação Pré-Clínica de Medicamentos , Técnicas Eletroquímicas , Ensaios de Triagem em Larga Escala , Peptídeos/síntese química , Processos Fotoquímicos , Técnicas de Síntese em Fase Sólida
8.
Ann Endocrinol (Paris) ; 80(2): 128-133, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30833018

RESUMO

In the modern world, type-2 diabetes mellitus has become a leading public healthcare problem, due to major risks of morbidity and mortality. Prevalence has increased significantly in recent decades. Treatment involves oral hypoglycemic agents or insulin replacement therapy. Development is ongoing for cell-based diabetes therapies using stem cells with the potential to differentiate into insulin-producing cells (IPCs): embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and stem cells from adult pancreas, liver, central nervous system, bone marrow and adipose tissue. Successful induction of iPSCs, however, depends on the quantity and quality of available stem cells and the development of adapted protocols determining the environment of extrinsic factors and involvement of small molecules. Validating such new cell therapies must be founded on this experimental rationale.


Assuntos
Fatores Biológicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células-Tronco Pluripotentes/efeitos dos fármacos , Animais , Fatores Biológicos/análise , Fatores Biológicos/isolamento & purificação , Técnicas de Cultura de Células , Reprogramação Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Insulina/metabolismo , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Pâncreas/citologia , Pâncreas/efeitos dos fármacos , Pâncreas/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Bibliotecas de Moléculas Pequenas/análise
9.
Org Biomol Chem ; 17(11): 3010-3017, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30816385

RESUMO

"Minimalist" small molecule tagging (MSMT) is a promising approach that easily converts bioactive compounds into affinity-based probes (AfBPs) for proteomic studies. In this work, seven bioactive compounds targeting diversified protein classes were installed with "minimalist" linkers through common reactions to generate the corresponding AfBPs. These probes were evaluated for cell-based protein profiling and target validation. Among them, the entinostat-derived probe EN and the camptothecin-derived probe CA were further utilized in cellular imaging and SILAC-based large-scale target identification. Our extensive studies suggest that the "minimalist" small molecule tagging approach could be expanded to different classes of bioactive compounds for modification into AfBPs as a dual functional tool for both proteomics and cellular imaging.


Assuntos
Camptotecina/análise , Camptotecina/química , Proteínas de Neoplasias/análise , Imagem Óptica , Proteômica , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/química , Camptotecina/síntese química , Células Hep G2 , Humanos , Proteínas Recombinantes/análise , Bibliotecas de Moléculas Pequenas/síntese química
10.
Cell ; 176(4): 687-701.e5, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30735632

RESUMO

Female Aedes aegypti mosquitoes bite humans to obtain blood to develop their eggs. Remarkably, their strong attraction to humans is suppressed for days after the blood meal by an unknown mechanism. We investigated a role for neuropeptide Y (NPY)-related signaling in long-term behavioral suppression and discovered that drugs targeting human NPY receptors modulate mosquito host-seeking. In a screen of all 49 predicted Ae. aegypti peptide receptors, we identified NPY-like receptor 7 (NPYLR7) as the sole target of these drugs. To obtain small-molecule agonists selective for NPYLR7, we performed a high-throughput cell-based assay of 265,211 compounds and isolated six highly selective NPYLR7 agonists that inhibit mosquito attraction to humans. NPYLR7 CRISPR-Cas9 null mutants are defective in behavioral suppression and resistant to these drugs. Finally, we show that these drugs can inhibit biting and blood-feeding on a live host, suggesting a novel approach to control infectious disease transmission by controlling mosquito behavior. VIDEO ABSTRACT.


Assuntos
Comportamento de Busca por Hospedeiro/efeitos dos fármacos , Mosquitos Vetores/efeitos dos fármacos , Receptores de Neuropeptídeo Y/agonistas , Aedes/metabolismo , Animais , Comportamento Alimentar/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Mordeduras e Picadas de Insetos , Receptores de Neuropeptídeo Y/metabolismo , Bibliotecas de Moléculas Pequenas/análise
11.
PLoS One ; 14(1): e0210525, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30625228

RESUMO

Werner syndrome (WS), an autosomal recessive genetic disorder, displays accelerated clinical symptoms of aging leading to a mean lifespan less than 50 years. The WS helicase-nuclease (WRN) is involved in many important pathways including DNA replication, recombination and repair. Replicating cells are dependent on helicase activity, leading to the pursuit of human helicases as potential therapeutic targets for cancer treatment. Small molecule inhibitors of DNA helicases can be used to induce synthetic lethality, which attempts to target helicase-dependent compensatory DNA repair pathways in tumor cells that are already genetically deficient in a specific pathway of DNA repair. Alternatively, helicase inhibitors may be useful as tools to study the specialized roles of helicases in replication and DNA repair. In this study, approximately 350,000 small molecules were screened based on their ability to inhibit duplex DNA unwinding by a catalytically active WRN helicase domain fragment in a high-throughput fluorometric assay to discover new non-covalent small molecule inhibitors of the WRN helicase. Select compounds were screened to exclude ones that inhibited DNA unwinding by other helicases in the screen, bound non-specifically to DNA, acted as irreversible inhibitors, or possessed unfavorable chemical properties. Several compounds were tested for their ability to impair proliferation of cultured tumor cells. We observed that two of the newly identified WRN helicase inhibitors inhibited proliferation of cancer cells in a lineage-dependent manner. These studies represent the first high-throughput screen for WRN helicase inhibitors and the results have implications for anti-cancer strategies targeting WRN in different cancer cells and genetic backgrounds.


Assuntos
Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Helicase da Síndrome de Werner/antagonistas & inibidores , Biocatálise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Fluorometria , Humanos , Concentração Inibidora 50 , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/química , Helicase da Síndrome de Werner/metabolismo
12.
Int J Mol Med ; 43(1): 276-284, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30431066

RESUMO

The objectives of the present study comprised the recognition of major genes related to pulmonary thromboembolism (PTE) and the evaluation of their functional enrichment levels, in addition to the identification of small chemical molecules that may offer potential for use in PTE treatment. The RNA expression profiling of GSE84738 was obtained from the Gene Expression Omnibus database. Following data preprocessing, the differently expressed genes (DEGs) between the PTE group and the control group were identified using the Linear Models for Microarray package. Subsequently, the protein­protein interaction (PPI) network of these DEGs was examined using the Search Tool for the Retrieval of Interacting Genes/Proteins database, visualized via Cytoscape. The most significantly clustered modules in the network were identified using Multi Contrast Delayed Enhancement, a plugin of Cytoscape. Subsequently, functional enrichment analysis of the DEGs was performed, using the Database for Annotation Visualization and Integrated Discovery tool. Furthermore, the chemical­target interaction networks were investigated using the Comparative Toxicogenomics Database as visualized via Cytoscape. A total of 548 DEGs (262 upregulated and 286 downregulated) were identified in the PTE group, compared with the control group. The upregulated and downregulated genes were enriched in Gene Ontology terms related to inflammation and eye sarcolemma, respectively. Tumor necrosis factor (TNF) and erb­b2 receptor tyrosine kinase 2 (ERBB2) were upregulated genes that ranked higher in the PPI network (47 and 40 degrees, respectively) whereas C­JUN was the most downregulated gene (46). Small chemical molecules ethinyl (135), cyclosporine (126), thrombomodulin precursor (113) and tretinoin (111) had >100 degrees in the DEG­chemical interaction network. In addition, ethinyl targeted to TNF, whereas TNF and ERBB2 were targeted by cyclosporine, and tretinoin was a targeted chemical of ERBB2. Therefore, cyclosporine, ethinyl, and tretinoin may be potential targets for PTE treatment.


Assuntos
Biologia Computacional , Embolia Pulmonar/tratamento farmacológico , Embolia Pulmonar/genética , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/uso terapêutico , Animais , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Ontologia Genética , Genoma , Mapas de Interação de Proteínas/genética , Coelhos , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação para Cima/genética
13.
Methods Mol Biol ; 1906: 197-206, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30488394

RESUMO

Microchip electrophoresis (ME) results from miniaturization of capillary electrophoresis (CE) to a microfabricated separation device. Both techniques have common characteristics, but in some aspects, the microfluidic separation device has unique features resulting from its planar miniaturized format. Here we describe the process to transfer of CE to ME and the benefits and drawbacks of the chip with respect to the capillary. A practical guide for method development on the microchip for small ionizable molecules such as phenolic compounds, amino acids, or alkaloids is also presented.


Assuntos
Eletroforese em Microchip/instrumentação , Bibliotecas de Moléculas Pequenas/análise , Alcaloides/análise , Aminoácidos/análise , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/instrumentação , Fenóis/análise
14.
Biomed Chromatogr ; 33(3): e4430, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30412644

RESUMO

Bioanalysis plays a key role during the drug discovery process to generate the pharmacokinetic data to facilitate unbiased evaluation of leads, optimized leads and drug candidates. Such pharmacokinetic data are used to enable key decisions in the drug discovery process. The aim of the work is to put forward a new strategy of performing the incurred sample reanalysis for select small molecule novel chemical entities at different stages of drug discovery prior to candidate selection. Three discovery programs representing hits, leads and optimized lead candidates were selected for the incurred sample reanalysis (ISR) analysis. From each discovery program, two novel chemical entities were selected for the ISR analysis. The time points considered for ISR generally varied among the programs; however, samples coinciding with drug absorption, distribution and elimination were considered in the ISR assessment. With the exception of a single ISR value that gave a high deviation (about 63%), the observed ISR values supported the discovery bioanalytical assays. While the individual bioanalytical laboratory can draw an algorithm for selecting novel chemical entities and fixing the acceptance criteria for the ISR data, it is proposed that the percentage difference between ISR vs. original concentration for 67% of the repeat samples is contained within ±30% for discovery bioanalysis.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Descoberta de Drogas/métodos , Descoberta de Drogas/normas , Espectrometria de Massas/métodos , Animais , Drogas em Investigação/análise , Drogas em Investigação/farmacocinética , Feminino , Masculino , Camundongos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacocinética
15.
Methods Mol Biol ; 1869: 189-196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30324524

RESUMO

Traditionally anti-cancer therapeutics have been designed to target rapidly proliferating cells causing DNA damage and inducing apoptosis. However, with the development of the cancer stem cell (CSC) hypothesis, it has been postulated that a rare, slow dividing tumor cell population is able to escape therapy and contribute to tumor relapse and metastasis. The advances in characterization of CSCs across multiple cancer subtypes have allowed for development of targeted therapies using small molecule inhibitors. In this chapter, we describe two in vitro assays measuring proliferation and secondary sphere formation, which have become gold-standard assays to evaluate the effects of targeted therapies against CSCs. Together these assays constitute a rapid, inexpensive, and highly reproducible pipeline for testing small molecule inhibitors prior to more resource demanding in vivo studies.


Assuntos
Bioensaio/métodos , Bibliotecas de Moléculas Pequenas/análise , Linhagem Celular , Proliferação de Células , Autorrenovação Celular , Humanos , Concentração Inibidora 50 , Suspensões
16.
Curr Med Chem ; 26(21): 3958-4002, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-28462709

RESUMO

In recent years, fluorescent probes have recently attracted attention from researchers. As a vital trace metal element, Cu2+ has an important role in the human body and environment. Therefore, the development and design of Cu2+ small-molecular fluorescent probes has been an active research area. This review focuses on the developments in the area of small-molecular fluorescent probes for Cu2+ in biological applications according to different sensing mechanisms including charge transfer (CT), electron transfer, energy transfer, excited-state intramolecular proton transfer (ESIPT).


Assuntos
Cobre/análise , Cobre/metabolismo , Corantes Fluorescentes/análise , Imagem Óptica/métodos , Bibliotecas de Moléculas Pequenas/análise , Transporte de Elétrons , Transferência de Energia , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Prótons , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química
17.
Anal Chem ; 90(24): 14347-14354, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30411873

RESUMO

Aptamers are recognized as competitive affinity reagents; their application, however, often suffers from their relatively low target binding affinity, especially for small molecules. We herein introduce the concept of a recognition-enhanced metastably shielded aptamer probe (RMSApt) and explore its performance for digital quantification of low-affinity small molecules. The RMSApt design employs the idea of constructing an allosteric aptamer probe conferring a minor energy gap in the recognition switch process to facilitate target binding and probe response, in turn significantly improving the recognition efficiency for low-affinity targets. The probe design strategy boosts the application of aptamers for precisely quantifying targets with a dissociation constant Kd ranging from 10-4 to 10-9 M, which would cover most of the small-molecule species that exist binding aptamers. Thus, RMSApt would facilitate the translation of aptamers for medical diagnosis, food safety, and environmental screening.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Bibliotecas de Moléculas Pequenas/análise , Imagem Óptica , Bibliotecas de Moléculas Pequenas/metabolismo
18.
Nat Protoc ; 13(12): 2827-2843, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30382243

RESUMO

In vitro models of the blood-brain barrier (BBB) are critical tools for the study of BBB transport and the development of drugs that can reach the CNS. Brain endothelial cells grown in culture are often used to model the BBB; however, it is challenging to maintain reproducible BBB properties and function. 'BBB organoids' are obtained following coculture of endothelial cells, pericytes and astrocytes under low-adhesion conditions. These organoids reproduce many features of the BBB, including the expression of tight junctions, molecular transporters and drug efflux pumps, and hence can be used to model drug transport across the BBB. This protocol provides a comprehensive description of the techniques required to culture and maintain BBB organoids. We also describe two separate detection approaches that can be used to analyze drug penetration into the organoids: confocal fluorescence microscopy and mass spectrometry imaging. Using our protocol, BBB organoids can be established within 2-3 d. An additional day is required to analyze drug permeability. The BBB organoid platform represents an accurate, versatile and cost-effective in vitro tool. It can easily be scaled to a high-throughput format, offering a tool for BBB modeling that could accelerate therapeutic discovery for the treatment of various neuropathologies.


Assuntos
Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Técnicas de Cocultura/métodos , Células Endoteliais/metabolismo , Organoides/metabolismo , Pericitos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacocinética , Astrócitos/citologia , Transporte Biológico , Barreira Hematoencefálica/citologia , Linhagem Celular , Células Endoteliais/citologia , Humanos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Organoides/citologia , Pericitos/citologia , Permeabilidade , Preparações Farmacêuticas/análise , Farmacocinética , Bibliotecas de Moléculas Pequenas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
19.
Anal Chim Acta ; 1034: 161-167, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30193630

RESUMO

An ultrasensitive lateral-flow assay is developed for rapid quantitative detection of small molecules on-site. The conceptual novelty, which transfers lateral-flow assays to the category of highly sensitive quantitative systems, is due to employment of a bifunctional ligand combined with volumetric registration of magnetic nanolabels. The ligand provides extremely high affinity for trapping the nanolabels and, simultaneously, efficiently competes with the analyzed molecules for the limited quantity of antigen-binding sites on the nanolabels. The developed assay has been demonstrated as the first express method for measuring in human serum of free thyroxine (fT4). The limit of detection is 20 fМ or 16 fg/ml at the assay time <30 min with the dynamic range of 3 orders. Besides, we present the results of first characterization of kinetic parameters of interaction between free thyroxine and monoclonal antibody, as well as of competitive relationship between fT4 and fT4-biotin. The proposed universal platform can be used for ultrasensitive detection of small molecules in human in vitro diagnostics, veterinary, biosafety and counter-terrorism, food quality control, environmental monitoring, etc., as well as for search of new, previously undetectable, diagnostic markers in medicine.


Assuntos
Cromatografia de Afinidade , Nanopartículas de Magnetita/química , Bibliotecas de Moléculas Pequenas/análise , Tiroxina/sangue , Anticorpos Monoclonais/imunologia , Biotina/química , Humanos , Ligantes , Bibliotecas de Moléculas Pequenas/química , Tiroxina/química , Tiroxina/imunologia
20.
Chem Rev ; 118(20): 10617-10625, 2018 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-30247025

RESUMO

Plasmonic biosensing has been used for fast, real-time, and label-free probing of biologically relevant analytes, where the main challenges are to detect small molecules at ultralow concentrations and produce compact devices for point-of-care (PoC) analysis. This review discusses the most recent, or even emerging, trends in plasmonic biosensing, with novel platforms which exploit unique physicochemical properties and versatility of new materials. In addition to the well-established use of localized surface plasmon resonance (LSPR), three major areas have been identified in these new trends: chiral plasmonics, magnetoplasmonics, and quantum plasmonics. In describing the recent advances, emphasis is placed on the design and manufacture of portable devices working with low loss in different frequency ranges, from the infrared to the visible.


Assuntos
Bibliotecas de Moléculas Pequenas/análise , Ressonância de Plasmônio de Superfície , Sistemas Automatizados de Assistência Junto ao Leito
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