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1.
Nat Commun ; 12(1): 4669, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344873

RESUMO

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.


Assuntos
Sistema Nervoso Central/metabolismo , Chaperonas Moleculares/metabolismo , Mapeamento de Interação de Proteínas/instrumentação , Proteoma/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Sondas Moleculares/farmacologia , Sondas Moleculares/uso terapêutico , Tomografia por Emissão de Pósitrons
2.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201632

RESUMO

The rapid development of super-resolution microscopy (SRM) techniques opens new avenues to examine cell and tissue details at a nanometer scale. Due to compatibility with specific labelling approaches, in vivo imaging and the relative ease of sample preparation, SRM appears to be a valuable alternative to laborious electron microscopy techniques. SRM, however, is not free from drawbacks, with the rapid quenching of the fluorescence signal, sensitivity to spherical aberrations and light scattering that typically limits imaging depth up to few micrometers being the most pronounced ones. Recently presented and robustly optimized sets of tissue optical clearing (TOC) techniques turn biological specimens transparent, which greatly increases the tissue thickness that is available for imaging without loss of resolution. Hence, SRM and TOC are naturally synergistic techniques, and a proper combination of these might promptly reveal the three-dimensional structure of entire organs with nanometer resolution. As such, an effort to introduce large-scale volumetric SRM has already started; in this review, we discuss TOC approaches that might be favorable during the preparation of SRM samples. Thus, special emphasis is put on TOC methods that enhance the preservation of fluorescence intensity, offer the homogenous distribution of molecular probes, and vastly decrease spherical aberrations. Finally, we review examples of studies in which both SRM and TOC were successfully applied to study biological systems.


Assuntos
Microscopia/métodos , Imagem Óptica/métodos , Animais , Encéfalo/diagnóstico por imagem , Fluorescência , Corantes Fluorescentes/química , Humanos , Processamento de Imagem Assistida por Computador/métodos , Sondas Moleculares/química , Fixação de Tecidos/métodos
3.
Nat Commun ; 12(1): 4518, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312393

RESUMO

Multiplexed optical imaging provides holistic visualization on a vast number of molecular targets, which has become increasingly essential for understanding complex biological processes and interactions. Vibrational microscopy has great potential owing to the sharp linewidth of vibrational spectra. In 2017, we demonstrated the coupling between electronic pre-resonant stimulated Raman scattering (epr-SRS) microscopy with a proposed library of 9-cyanopyronin-based dyes, named Manhattan Raman Scattering (MARS). Herein, we develop robust synthetic methodology to build MARS probes with different core atoms, expansion ring numbers, and stable isotope substitutions. We discover a predictive model to correlate their vibrational frequencies with structures, which guides rational design of MARS dyes with desirable Raman shifts. An expanded library of MARS probes with diverse functionalities is constructed. When coupled with epr-SRS microscopy, these MARS probes allow us to demonstrate not only many versatile labeling modalities but also increased multiplexing capacity. Hence, this work opens up next-generation vibrational imaging with greater utilities.


Assuntos
Corantes/química , Sondas Moleculares/química , Microscopia Óptica não Linear/métodos , Imagem Óptica/métodos , Pironina/química , Corantes/síntese química , Células HeLa , Humanos , Modelos Químicos , Sondas Moleculares/síntese química , Estrutura Molecular , Pironina/análogos & derivados , Pironina/síntese química , Análise Espectral Raman/métodos , Vibração
4.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067989

RESUMO

Magnetic resonance imaging (MRI) is often used to diagnose diseases due to its high spatial, temporal and soft tissue resolution. Frequently, probes or contrast agents are used to enhance the contrast in MRI to improve diagnostic accuracy. With the development of molecular imaging techniques, molecular MRI can be used to obtain 3D anatomical structure, physiology, pathology, and other relevant information regarding the lesion, which can provide an important reference for the accurate diagnosis and treatment of the disease in the early stages. Among existing contrast agents, smart or activatable nanoprobes can respond to selective stimuli, such as proving the presence of acidic pH, active enzymes, or reducing environments. The recently developed environment-responsive or smart MRI nanoprobes can specifically target cells based on differences in the cellular environment and improve the contrast between diseased tissues and normal tissues. Here, we review the design and application of these environment-responsive MRI nanoprobes.


Assuntos
Meios de Contraste/química , Imageamento por Ressonância Magnética/métodos , Imageamento por Ressonância Magnética/normas , Nanopartículas de Magnetita/química , Sondas Moleculares/química , Humanos
5.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068759

RESUMO

Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable 'turn-on' fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.


Assuntos
Benzotiazóis/química , DNA/análise , Proteínas/análise , Quinolinas/química , DNA/química , Fluorescência , Íons , Sondas Moleculares/química
6.
Chem Commun (Camb) ; 57(54): 6608-6611, 2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34114574

RESUMO

We report a rational strategy to deliberately construct the first asymmetric tetraarylimidazole-based AIE probe, integrating AIE behavior in synergy with ESIPT character to image endogenous LAP for the first time. It offered good sensitivity and selectivity, and concomitantly, was applied successfully for real-time tracking of LAP in the cisplatin-induced liver injury zebrafish model.


Assuntos
Imidazóis/química , Leucil Aminopeptidase/metabolismo , Animais , Leucil Aminopeptidase/química , Fígado/metabolismo , Sondas Moleculares/química , Peixe-Zebra
7.
Chem Commun (Camb) ; 57(51): 6249-6252, 2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34059853

RESUMO

A hydrophilic probe is employed to enrich exosomes from three kinds of cancer cells by TiO2-phosphate interaction and exosomal glycoproteins by hydrophilic interaction in succession. The probe performs efficiently in both the enrichment processes. And the analytical results confirm that unique exosomal glycoproteins can distinguish parent exosomes from others.


Assuntos
Exossomos/metabolismo , Glicoproteínas/análise , Sondas Moleculares/metabolismo , Linhagem Celular Tumoral , Óxido Ferroso-Férrico/química , Glutationa/química , Glutationa/metabolismo , Glicoproteínas/metabolismo , Glicosilação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanopartículas de Magnetita/química , Sondas Moleculares/química , Proteômica/métodos , Titânio/química
8.
Chem Commun (Camb) ; 57(52): 6392-6395, 2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34085079

RESUMO

Small molecules are frequently used as dyes, labels and markers to visualize and probe biophysical processes within cells. However, very little is generally known about the light-driven excited-state reactivity of such systems when placed in cells. Here an experimental approach to study ps time-resolved excited state dynamics of a benchmark molecular marker, astaxanthin, in live human cells is introduced.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Dimetil Sulfóxido/química , Humanos , Cinética , Células MCF-7 , Sondas Moleculares/química , Teoria Quântica , Espectrofotometria , Xantofilas/química , Xantofilas/farmacologia
9.
Nat Commun ; 12(1): 3435, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103511

RESUMO

To understand the underlying mechanisms of progressive neurophysiological phenomena, neural interfaces should interact bi-directionally with brain circuits over extended periods of time. However, such interfaces remain limited by the foreign body response that stems from the chemo-mechanical mismatch between the probes and the neural tissues. To address this challenge, we developed a multifunctional sensing and actuation platform consisting of multimaterial fibers intimately integrated within a soft hydrogel matrix mimicking the brain tissue. These hybrid devices possess adaptive bending stiffness determined by the hydration states of the hydrogel matrix. This enables their direct insertion into the deep brain regions, while minimizing tissue damage associated with the brain micromotion after implantation. The hydrogel hybrid devices permit electrophysiological, optogenetic, and behavioral studies of neural circuits with minimal foreign body responses and tracking of stable isolated single neuron potentials in freely moving mice over 6 months following implantation.


Assuntos
Técnicas Biossensoriais , Hidrogéis/química , Sondas Moleculares/química , Neurônios/fisiologia , Potenciais de Ação/fisiologia , Animais , Comportamento Animal , Bioensaio , Encéfalo/fisiologia , Fenômenos Eletrofisiológicos , Reação a Corpo Estranho/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Optogenética , Estresse Mecânico , Fatores de Tempo
10.
Int J Mol Sci ; 22(6)2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33804798

RESUMO

Sialidase cleaves sialic acid residues from glycans such as glycoproteins and glycolipids. In the brain, desorption of the sialic acid by sialidase is essential for synaptic plasticity, learning and memory and synaptic transmission. BTP3-Neu5Ac has been developed for sensitive imaging of sialidase enzyme activity in mammalian tissues. Sialidase activity in the rat hippocampus detected with BTP3-Neu5Ac increases rapidly by neuronal depolarization. It is presumed that an increased sialidase activity in conjunction with neural excitation is involved in the formation of the neural circuit for memory. Since sialidase inhibits the exocytosis of the excitatory neurotransmitter glutamate, the increased sialidase activity by neural excitation might play a role in the negative feedback mechanism against the glutamate release. Mammalian tissues other than the brain have also been stained with BTP3-Neu5Ac. On the basis of information on the sialidase activity imaging in the pancreas, it was found that sialidase inhibitor can be used as an anti-diabetic drug that can avoid hypoglycemia, a serious side effect of insulin secretagogues. In this review, we discuss the role of sialidase in the brain as well as in the pancreas and skin, as revealed by using a sialidase activity imaging probe. We also present the detection of influenza virus with BTP3-Neu5Ac and modification of BTP3-Neu5Ac.


Assuntos
Imagem Molecular , Sondas Moleculares , Neuraminidase/metabolismo , Animais , Meios de Contraste , Ativação Enzimática , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ácido Glutâmico/biossíntese , Humanos , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Neurônios/metabolismo , Imagem Óptica/métodos , Especificidade de Órgãos , Viroses/diagnóstico por imagem , Viroses/metabolismo , Viroses/virologia
11.
Nat Commun ; 12(1): 2385, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888701

RESUMO

Nanoparticle internalisation is crucial for the precise delivery of drug/genes to its intracellular targets. Conventional quantification strategies can provide the overall profiling of nanoparticle biodistribution, but fail to unambiguously differentiate the intracellularly bioavailable particles from those in tumour intravascular and extracellular microenvironment. Herein, we develop a binary ratiometric nanoreporter (BiRN) that can specifically convert subtle pH variations involved in the endocytic events into digitised signal output, enabling the accurately quantifying of cellular internalisation without introducing extracellular contributions. Using BiRN technology, we find only 10.7-28.2% of accumulated nanoparticles are internalised into intracellular compartments with high heterogeneity within and between different tumour types. We demonstrate the therapeutic responses of nanomedicines are successfully predicted based on intracellular nanoparticle exposure rather than the overall accumulation in tumour mass. This nonlinear optical nanotechnology offers a valuable imaging tool to evaluate the tumour targeting of new nanomedicines and stratify patients for personalised cancer therapy.


Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/análise , Imagem Molecular/métodos , Nanopartículas/análise , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Corantes Fluorescentes/química , Humanos , Microscopia Intravital , Camundongos , Sondas Moleculares/administração & dosagem , Sondas Moleculares/análise , Sondas Moleculares/química , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Imagem Óptica/métodos , Paclitaxel/administração & dosagem , Seleção de Pacientes , Reprodutibilidade dos Testes , Nanomedicina Teranóstica/métodos , Distribuição Tecidual , Microambiente Tumoral/efeitos dos fármacos
12.
J Med Chem ; 64(8): 4396-4409, 2021 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-33821652

RESUMO

Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.


Assuntos
Endotélio/química , Análise Espectral Raman , Vasos Sanguíneos/química , Vasos Sanguíneos/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Endotélio/metabolismo , Endotélio/fisiopatologia , Humanos , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Mitocôndrias/química , Mitocôndrias/metabolismo , Sondas Moleculares/química , Sondas Moleculares/metabolismo
13.
Molecules ; 26(7)2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33807474

RESUMO

Protein kinases are a large class of enzymes with numerous biological roles and many have been implicated in a vast array of diseases, including cancer and the novel coronavirus infection COVID-19. Thus, the development of chemical probes to selectively target each kinase is of great interest. Inhibition of protein kinases with ATP-competitive inhibitors has historically been the most widely used method. However, due to the highly conserved structures of ATP-sites, the identification of truly selective chemical probes is challenging. In this review, we use the Ser/Thr kinase CK2 as an example to highlight the historical challenges in effective and selective chemical probe development, alongside recent advances in the field and alternative strategies aiming to overcome these problems. The methods utilised for CK2 can be applied to an array of protein kinases to aid in the discovery of chemical probes to further understand each kinase's biology, with wide-reaching implications for drug development.


Assuntos
Caseína Quinase II/metabolismo , Sondas Moleculares/química , Inibidores de Proteínas Quinases/química , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , COVID-19 , Caseína Quinase II/química , Diclororribofuranosilbenzimidazol/química , Diclororribofuranosilbenzimidazol/farmacologia , Humanos , Sondas Moleculares/metabolismo , Naftiridinas/química , Naftiridinas/farmacologia , Fenazinas/química , Fenazinas/farmacologia , Polifenóis/química , Polifenóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia
14.
Anal Bioanal Chem ; 413(11): 2951-2960, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33765221

RESUMO

Quantitative characterization of Cr3+, an important element revealing human metabolism and biological environmental variation, is still difficult to achieve by conventional biochemical methods due to the lack of high-sensitivity, real-time techniques with rapid response detection. Using surface-enhanced Raman scattering (SERS), we construct an Au/Ag composite-based SERS nanoprobe for the quantitative characterization of Cr3+ content in solution, in which DL-mercaptosuccinic acid (DL-MSA) is employed for Raman signal enhancement, and 4-mercaptobenzoic acid (4-MBA) is chosen as the Raman reporter. The achieved result demonstrates obvious advantages of the synthesized Au/Ag composite-based SERS nanoprobe in sensitivity and response speed. Importantly, this Au/Ag composite-based SERS nanoprobe might provide a new strategy for dynamic monitoring of Cr3+ content in human metabolism.


Assuntos
Cromo/análise , Ouro/química , Nanopartículas Metálicas/química , Sondas Moleculares/química , Prata/química , Análise Espectral Raman/métodos , Células HEK293 , Humanos , Microscopia Eletrônica de Transmissão , Soluções , Espectrofotometria Ultravioleta
15.
Bioorg Med Chem Lett ; 40: 127903, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33713779

RESUMO

Folate and related derivatives are essential small molecules required for survival. Of significant interest is the biological role and necessity of folate in the crosstalk between commensal organisms and their respective hosts, including the tremendously complex human distal gut microbiome. Here, we designed a folate-based probe consisting of a photo-crosslinker to detect and quantitate folate-binding proteins from proteomic samples. We demonstrate the selectivity of our probe for the well-established human folate-binding protein dihydrofolate reductase and show no promiscuous labeling occurs with human caspase-3 or bovine serum albumin, which served as negative controls. Affinity-based enrichment of folate-binding proteins from an E. coli lysate in combination with mass spectrometry proteomics verified the ability of our probe to isolate low-abundance folate-dependent proteins. We envision that our probe will serve as a tool to elucidate the roles of commensal microbial folate-binding proteins in health and microbiome-related diseases.


Assuntos
Reagentes para Ligações Cruzadas/química , Transportadores de Ácido Fólico/análise , Ácido Fólico/química , Sondas Moleculares/química , Caspase 3/química , Cromatografia Líquida de Alta Pressão , Escherichia coli/química , Humanos , Microbiota/fisiologia , Processos Fotoquímicos , Proteômica , Soroalbumina Bovina/metabolismo , Espectrometria de Massas em Tandem , Tetra-Hidrofolato Desidrogenase/química
16.
Bioconjug Chem ; 32(4): 801-809, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33711232

RESUMO

Magnetic nanoparticles have been widely used for the separation of biomolecules for biological applications due to the mild and efficient separation process. In previous studies, core-shell magnetic nanoparticles (NPs) were designed for DNA extraction without much sequence specificity. In this work, to achieve highly selective DNA extraction, we designed a core-shell magnetic structure by coating polydopamine (PDA) on Fe3O4 NPs. Without divalent metal ions, PDA does not adsorb DNA at neutral pH. The Fe3O4@PDA NPs were then functionalized with spherical nucleic acids (SNA) to provide a high density of probe DNA. Fe3O4@PDA@SNA was also compared with when a linear SH-DNA was covalently attached to the NPs surface, showing a higher density of the probe SNA than SH-DNA can be loaded on the NPs in a remarkably shorter time. Nonspecific DNA extraction was thoroughly inhibited by both probes. DNA extraction by the Fe3O4@PDA@SNA was more effective as well as 5-fold faster than by the Fe3O4@PDA@SH-DNA, probably due to the favorable standing conformation of DNA strands in SNA. Moreover, extraction by Fe3O4@PDA@SNA showed high robustness in fetal bovine serum, and the same design can be used for selective detection of DNA. Finally, the method was also demonstrated on silica NPs and WS2 nanosheets for coating with PDA and SNA. Altogether, our findings revealed an interesting and general surface modification strategy using PDA@SNA conjugates for sequence-specific DNA extraction.


Assuntos
DNA/análise , DNA/isolamento & purificação , Indóis/química , Nanopartículas de Magnetita/química , Ácidos Nucleicos/química , Polímeros/química , Adsorção , Microscopia Eletrônica de Transmissão , Sondas Moleculares/química
17.
Org Biomol Chem ; 19(13): 2968-2977, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33729259

RESUMO

GH29 α-l-fucosidases catalyze hydrolysis of terminal α-l-fucosyl linkages with varying specificity and are expressed by prominent members of the human gut microbiota. Both homeostasis and dysbiosis at the human intestinal microbiota interface have been correlated with altered fucosidase activity. Herein we describe the development of a 2-deoxy-2-fluoro fucosyl fluoride derivative with an azide mini-tag as an activity-based probe (ABP) for selective in vitro labelling of GH29 α-l-fucosidases. Only catalytically active fucosidases are inactivated by this ABP, allowing their functionalization with a biotin reporter group via the CuAAC reaction and subsequent in-gel detection at nanogram levels. The ABP we present here is shown to be active against a GH29 α-l-fucosidase from Bacteroides fragilis and capable of labeling two other GH29 α-l-fucosidases with different linkage specificity, illustrating its broader utility. This novel ABP is a valuable addition to the toolbox of fucosidase probes by allowing identification and functional studies of the wide variety of GH29 fucosidases, including those in the gut microbiota.


Assuntos
Fucose/química , Sondas Moleculares/química , alfa-L-Fucosidase/análise , Bacteroides fragilis/enzimologia , Fucose/análogos & derivados , Fucose/farmacologia , Microbioma Gastrointestinal , Humanos , Sondas Moleculares/síntese química , Sondas Moleculares/farmacologia , Estrutura Molecular , alfa-L-Fucosidase/antagonistas & inibidores , alfa-L-Fucosidase/metabolismo
18.
ACS Chem Biol ; 16(4): 579-585, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33745272

RESUMO

Chemical probes are selective modulators that are used in cell assays to link a phenotype to a gene and have become indispensable tools to explore gene function and discover therapeutic targets. Chemical probe off-targets are a confounding factor as the observed phenotype may be driven by inhibition of an unknown off-target instead of the targeted protein. A negative control, a close chemical analog of the chemical probe that is inactive against the intended target, is typically used to verify that the phenotype is indeed driven by the targeted protein. Here, we compare the selectivity profiles of four unrelated chemical probes and their respective negative controls. We find that controls that chemically deviate from the probe by a single heavy atom can be inactive against up to 80% of known off-targets if the chemical modification has a charge-neutralizing effect. In such cases, a loss in phenotype upon treatment with the negative control may be driven by loss of inhibition of an off-target. To expand this analysis, we inspect the crystal structures of 90 pairs of unrelated proteins, where both proteins within each pair is in complex with the same drug-like ligand. We computationally estimate that in 50% of cases, methylation of the ligand (a simple chemical modification often used to generate negative controls) at a position that will preclude binding to one protein (the intended target) will also preclude binding to the other (the off-target). These results emphasize the need to select negative controls with care and profile both chemical probes and negative controls against diverse protein arrays to verify that off-targets of probes are also hit by negative controls. When available, a best practice should be to verify that two unrelated chemical probes targeting the same protein elicit the same phenotype.


Assuntos
Sondas Moleculares/química , Bibliotecas de Moléculas Pequenas/química , Bases de Dados de Proteínas , Humanos , Ligantes
19.
Int J Nanomedicine ; 16: 2311-2322, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33776435

RESUMO

Background: Alzheimer's disease (AD) is a neurodegenerative chronic disorder that causes dementia and problems in thinking, cognitive impairment and behavioral changes. Amyloid-beta (Aß) is a peptide involved in AD progression, and a high level of Aß is highly correlated with severe AD. Identifying and quantifying Aß levels helps in the early treatment of AD and reduces the factors associated with AD. Materials and Methods: This research introduced a dual probe detection system involving aptamers and antibodies to identify Aß. Aptamers and antibodies were attached to the gold (Au) urchin and hybrid on the carbon nanohorn-modified surface. The nanohorn was immobilized on the sensor surface by using an amine linker, and then a Au urchin dual probe was immobilized. Results: This dual probe-modified surface enhanced the current flow during Aß detection compared with the surface with antibody as the probe. This dual probe interacted with higher numbers of Aß peptides and reached the detection limit at 10 fM with R2=0.992. Furthermore, control experiments with nonimmune antibodies, complementary aptamer sequences and control proteins did not display the current responses, indicating the specific detection of Aß. Conclusion: Aß-spiked artificial cerebrospinal fluid showed a similar response to current changes, confirming the selective identification of Aß.


Assuntos
Doença de Alzheimer/diagnóstico , Ouro/química , Sondas Moleculares/química , Nanopartículas/química , Peptídeos beta-Amiloides/metabolismo , Eletrodos , Humanos , Limite de Detecção , Modelos Lineares , Nanopartículas/ultraestrutura , Fragmentos de Peptídeos , Multimerização Proteica , Reprodutibilidade dos Testes , Espectrometria por Raios X , Propriedades de Superfície
20.
Molecules ; 26(5)2021 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-33652775

RESUMO

Europium (III) luminescent chelates possess intrinsic photophysical properties that are extremely useful in a wide range of applications. The lack of examples of coumarin-based lanthanide complexes is mainly due to poor photo-sensitization attempts. However, with the appeal of using such a versatile scaffold as antenna, especially in the development of responsive molecular probes, it is worth the effort to research new structural motifs. In this work, we present a series of two new tris coumarin-dipicolinate europium (III) complexes, specifically tailored to be either a mono or a dual emitter, tuning their properties with a simple chemical modification. We also encountered a rich chemical speciation in solution, studied in detail by means of paramagnetic NMR and emission spectroscopy.


Assuntos
Complexos de Coordenação/química , Cumarínicos/química , Európio/química , Sondas Moleculares/química , Quelantes/química , Elementos da Série dos Lantanídeos/química , Luminescência , Espectroscopia de Ressonância Magnética
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