Small-molecule Probes Research Articles

Tuning the Cellular Uptake and Retention of Rhodamine Dyes by Molecular Engineering for High-Contrast Imaging of Cancer Cells.

Probes allowing high-contrast discrimination of cancer cells and effective retention are powerful tools for the early diagnosis and treatment of cancer. However, conventional small-molecule probes often show limited performance in both aspects. Herein, we report an ingenious molecular engineering strategy for tuning the cellular uptake and retention of rhodamine dyes. Introduction of polar aminoethyl leads to the increased brightness and reduced cellular uptake of dyes, and this change can be reversed by amino acetylation. Moreover, these modifications allow cancer cells to take up more dyes than normal cells (16-fold) through active transport. Specifically, we further improve the signal contrast (56-fold) between cancer and normal cells by constructing activatable probes and confirm that the released fluorophore can remain in cancer cells with extended time, enabling long-term and specific tumor imaging.

A Small-Molecule Fluorescence Probe for Nuclear ATP.

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1-3.3-fold and 3.9-7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.

Strategies for Competitive Activity‐Based Protein Profiling in Small Molecule Inhibitor Discovery and Characterization

AbstractSince its introduction by Cravatt and colleagues in 1999, activity‐based protein profiling (ABPP) has become widely utilized throughout academia, government, and industry laboratories to study enzymes spanning numerous gene families in a multitude of biological systems. As a variation of ABPP, competitive ABPP provides a powerful approach to characterize the binding behavior of small molecule probes and clinical drugs throughout the functional proteome. The power and flexibility of competitive ABPP are exemplified by a wide range of creative adaptions which increase assay throughput, enable diverse detection schemes, and support the implementation of this approach within a hybrid target‐based screening platform. We review major developments in competitive ABPP through a compare‐contrast format to provide a useful introduction to this enabling technology for scientists in chemical biology and drug discovery.

Cell-Surface Targeting of Fluorophores in Drosophila for Rapid Neuroanatomy Visualization.

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small-molecule tethering proteins, HaloTag-CD4 (Kirk et al. Front. Neurosci. 2021, 15, 754027) and SNAPf-CD4, which directly label transgene-expressing cells with commercially available ligand-substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines, which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live-brain tissues. Pan-neural expression of SNAPf-CD4 enabled the registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small-molecule probes, drugs, and actuators.

De novo design of a novel AIE fluorescent probe tailored to autophagy visualization via pH manipulation

BackgroundMacroautophagy is an essential cellular self-protection mechanism, and defective autophagy has been considered to contribute to a variety of diseases. During the process, cytoplasmic components are transported via autophagosomes to acidic lysosomes for metabolism and recycling, which represents application niches for lysosome-targeted fluorescent probes. Additionally, in view of the complexity of the autophagy pathway, it entails more stringent requirements for probes suitable for monitoring autophagy. Meanwhile, aggregation-induced emission (AIE) fluorescent probes have been impressively demonstrated in the biomedical field, which bring fascinating possibilities to the autophagy visualization.MethodsWe reported a generalizable de novo design of a novel pH-sensitive AIE probe ASMP-AP tailored to lysosome targeting for the interpretation of autophagy. Firstly, the theoretical calculation was carried out followed by the investigation of optical properties. Then, the performance of ASMP-AP in visualizing autophagy was corroborated by starvation or drugs treatments. Furthermore, the capability of ASMP-AP to monitor autophagy was demonstrated in ex vivo liver tissue and zebrafish in vivo.ResultsASMP-AP displays a large stokes shift, great cell permeability and good biocompatibility. More importantly, ASMP-AP enables a good linear response to pH, which derives from the fact that its aggregation state can be manipulated by the acidity. It was successfully applied for imaging autophagy in living cells and was proved capable of monitoring mitophagy. Moreover, this novel molecular tool was validated by ex vivo visualization of activated autophagy in drug-induced liver injury model. Interestingly, it provided a meaningful pharmacological insight that the melanin inhibitor 1-phenyl-2-thiourea (PTU)-induced autophagy was clearly presented in wild-type zebrafish.ConclusionsASMP-AP offers a simple yet effective tool for studying lysosome and autophagy. This is the first instance to visualize autophagy in zebrafish using a small-molecule probe with AIE characters, accurate lysosome targeting and simultaneous pH sensitivity. Ultimately, this novel fluorescent system has great potential for in vivo translation to fuel autophagy research.Graphical

Research Advances Through Activity‐Based Lipid Hydrolase Profiling

AbstractActivity‐based proteomic profiling (ABPP) enables the functional study of enzymes by employing small molecule probes that bind covalently to the active site of an enzyme. Activity‐based probes can penetrate cells and tissues and thereby allow enzymes to be targeted/labelled in their native state. Probes can be designed to target individual enzymes or whole enzyme groups, which makes ABPP a versatile protein profiling technique. In this review, we give an overview of research advances through ABPP in the context of lipid hydrolase research. We report of lipid hydrolases that were discovered and characterized through ABPP, and aim to give an overview of commonly used probes as well as inhibitors that were discovered and characterized by competitive ABPP. Lastly, this review aims to raise caveats and current limitations of this protein profiling technique.

Practical Guidance for Developing Small-Molecule Optical Probes for In Vivo Imaging.

The WMIS Education Committee (2019-2022) reached a consensus that white papers on molecular imaging could be beneficial for practitioners of molecular imaging at their early career stages and other scientists who are interested in molecular imaging. With this consensus, the committee plans to publish a series of white papers on topics related to the daily practice of molecular imaging. In this white paper, we aim to provide practical guidance that could be helpful for optical molecular imaging, particularly for small molecule probe development and validation in vitro and in vivo. The focus of this paper is preclinical animal studies with small-molecule optical probes. Near-infrared fluorescence imaging, bioluminescence imaging, chemiluminescence imaging, image-guided surgery, and Cerenkov luminescence imaging are discussed in this white paper.

Selective probes and antagonists for G protein-coupled receptor LPAR1.

The G protein-coupled receptors (GPCRs) constitute the largest class of signaling molecules in the human genome with diverse disease pathogenesis, yet many of receptors with unknown antagonists continue to remain. The complex biology and potential for drug discovery within this class provides strong incentives for chemical biology approaches that seek to develop small molecule probes. In addition to this, it will also help to facilitate the elucidation of mechanistic pathways and enable the specific manipulation of the individual receptors’ activity. We have initiated a small molecule antagonist that actively targets lysophosphatidic acid receptor (LPAR1), a clinically important target that is linked to several metabolic and inflammatory diseases including cancer, fibrosis, and rheumatoid arthritis. While numerous selective LPAR1 antagonists have been developed their clinical efficacy has only been evaluated in idiopathic pulmonary fibrosis. As a useful aid in the various efforts of identifying novel effective LPAR1 antagonists, diverse computational methods can serve as an important and valuable tool. In particular, the sequent in-silico approaches such as virtual screening, molecular docking, molecular dynamics simulation, and protein-ligand binding free energy are able to refine the accuracy of in-silico approaches of GPCR-antagonist complexes. In our study, we focused on trying to identify novel LPAR1 antagonists using the in-silico virtual screening of commercial databases on the human LPAR1 model. Various molecular modeling methodologies such as molecular docking and dynamics simulation, along with different drug likeness filtering criteria, were applied to select anti-LPAR1 antagonists as promising candidate molecules based on different known lead compounds. In-vitro binding assays of the selected molecules are expected to demonstrate activity within the nanomolar range of Ki or IC50. The current integrated computational and experimental methods used in this work can provide new insights for systematic hit identification for novel anti-LPAR1 agents.

A Blood-Responsive AIE Bioprobe for the Ultrasensitive Detection and Assessment of Subarachnoid Hemorrhage.

Subarachnoid hemorrhage (SAH) is a severe subtype of stroke caused by the rupturing of blood vessels in the brain. The ability to accurately assess the degree of bleeding in an SAH model is crucial for understanding the brain-damage mechanisms and developing therapeutic strategies. However, current methods are unable to monitor microbleeding owing to their limited sensitivities. Herein, a new bleeding assessment system using a bioprobe TTVP with aggregation-induced emission (AIE) characteristics is demonstrated. TTVP is a water-soluble, small-molecule probe that specifically interacts with blood. Taking advantage of its AIE characteristics, cell membranes affinity, and albumin-targeting ability, TTVP fluoresces in bleeding areas and detects the presence of blood with a high signal-to-noise (S/N) ratio. The degree of SAH bleeding in an endovascular perforation model is clearly evaluated based on the intensity of the fluorescence observed in the brain, which enables the ultrasensitive detection of mirco-bleeding in the SAH model in a manner that outperforms the current imaging strategies. This method serves as a promising tool for the sensitive analysis of the degree of bleeding in SAHs and other hemorrhagic diseases.

Two Color Imaging of Different Hypoxia Levels in Cancer Cells.

Hypoxia (low oxygen levels) occurs in a range of biological contexts, including plants, bacterial biofilms, and solid tumors; it elicits responses from these biological systems that impact their survival. For example, conditions of low oxygen make treating tumors more difficult and have a negative impact on patient prognosis. Therefore, chemical probes that enable the study of biological hypoxia are valuable tools to increase the understanding of disease-related conditions that involve low oxygen levels, ultimately leading to improved diagnosis and treatment. While small-molecule hypoxia-sensing probes exist, the majority of these image only very severe hypoxia (<1% O2) and therefore do not give a full picture of heterogeneous biological hypoxia. Commonly used antibody-based imaging tools for hypoxia are less convenient than small molecules, as secondary detection steps involving immunostaining are required. Here, we report the synthesis, electrochemical properties, photophysical analysis, and biological validation of a range of indolequinone-based bioreductive fluorescent probes. We show that these compounds image different levels of hypoxia in 2D and 3D cell cultures. The resorufin-based probe 2 was activated in conditions of 4% O2 and lower, while the Me-Tokyo Green-based probe 4 was only activated in severe hypoxia─0.5% O2 and less. Simultaneous application of these compounds in spheroids revealed that compound 2 images similar levels of hypoxia to pimonidazole, while compound 4 images more extreme hypoxia in a manner analogous to EF5. Compounds 2 and 4 are therefore useful tools to study hypoxia in a cellular setting and represent convenient alternatives to antibody-based imaging approaches.

68Ga-labeled amphiphilic polymer nanoparticles for PET imaging of sentinel lymph node metastasis.

Precise diagnosis of lymph node metastasis is important for therapeutic regimen planning, prognosis analysis and probably better outcomes for cancer patients. In this work, 68Ga-labeled amphiphilic alternating copolymers nanoparticles with different rigid ligands were synthesized as positron emission tomography (PET) probes for lymph node metastasis imaging. The labeling efficiency and stability of nanoparticles was improved with increased rigidity of coordination unit. PU(68Ga-L-MDI-PEG) nanoparticles (PU(68Ga-L-MDI-PEG) NPs) with the strongest rigidity of coordination unit exhibited the lowest critical micelle concentration, the best 68Ga labeling efficiency and stability. During in vivo lymph node metastasis imaging, PU(68Ga-L-MDI-PEG) NPs led to different accumulations in normal lymph nodes (N-LN) and tumor metastasized sentinel lymph nodes (T-SLN), which resulted in different PET signal presentation, making it feasible to differentiate N-LN from T-SLN. In comparison, small molecule probe 68GaL had poor lymph node accumulation, not only making it difficult to find lymph nodes on PET/computed tomography scan, but also tough to distinguish N-LN from metastatic ones. Overall, this work provides a reference for design of 68Ga labeled polymeric nanoparticles with high chelation efficiency and stability, as sensitive PET probes for lymph node imaging.

Small Molecule Probes for 19F Magnetic Resonance Imaging.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTSmall Molecule Probes for 19F Magnetic Resonance ImagingAo LiAo LiThe MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Fujian Provincial Key Laboratory of Chemical Biology, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen361005, ChinaMore by Ao LiView Biography, Xiangjie LuoXiangjie LuoThe MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Fujian Provincial Key Laboratory of Chemical Biology, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen361005, ChinaMore by Xiangjie LuoView Biography, Dongxia ChenDongxia ChenThe MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Fujian Provincial Key Laboratory of Chemical Biology, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen361005, ChinaMore by Dongxia ChenView Biography, Lingxuan LiLingxuan LiThe MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Fujian Provincial Key Laboratory of Chemical Biology, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen361005, ChinaMore by Lingxuan LiView Biography, Hongyu Lin*Hongyu LinThe MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Fujian Provincial Key Laboratory of Chemical Biology, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen361005, China*[email protected]More by Hongyu LinView Biographyhttps://orcid.org/0000-0002-5675-8537, and Jinhao Gao*Jinhao GaoThe MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, Fujian Provincial Key Laboratory of Chemical Biology, and Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen361005, China*[email protected]More by Jinhao GaoView Biographyhttps://orcid.org/0000-0003-3215-7013Cite this: Anal. Chem. 2023, 95, 1, 70–82Publication Date (Web):January 10, 2023Publication History Received14 October 2022Published online10 January 2023Published inissue 10 January 2023https://doi.org/10.1021/acs.analchem.2c04539Copyright © 2023 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views221Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (7 MB) Get e-AlertsSUBJECTS:Ions,Magnetic resonance imaging,Probes,Sensor probes,Small molecules Get e-Alerts

Sensitive imaging of Endoplasmic reticulum (ER) autophagy with an acidity-reporting ER-Tracker

ABSTRACT Macroautophagic/autophagic turnover of endoplasmic reticulum (reticulophagy) is critical for cell health. Herein we reported a sensitive fluorescence-on imaging of reticulophagy using a small molecule probe (ER-proRed) comprised of green-emissive fluorinated rhodol for ER targeting and nonfluorescent rhodamine-lactam prone to lysosome-triggered red fluorescence. Partitioned in ER to exhibit green fluorescence, ER-proRed gives intense red fluorescence upon co-delivery with ER into acidic lysosomes. Serving as the signal of reticulophagy, the turning on of red fluorescence enables discernment of reticulophagy induced by starvation, varied levels of reticulophagic receptors, and chemical agents such as etoposide and sodium butyrate. These results show ER probes optically activatable in lysosomes, such as ER-proRed, offer a sensitive and simplified tool for studying reticulophagy in biology and diseases. Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CQ, chloroquine diphosphate; ER, endoplasmic reticulum; FHR, fluorinated hydrophobic rhodol; GFP, green fluorescent protein; Reticulophagy, selective autophagy of ER; RFP, red fluorescent protein; ROX, X-rhodamine; UPR, unfolded protein response

Recent progress in the development of small-molecule double-locked logic gate fluorescence probes.

Various bioactive substances are simultaneously involved in physiological processes, and research on the synergistic effect of them can promote the study of pathological mechanisms. To achieve this purpose, several small-molecule double-locked logic gate fluorescence probes have been developed recently. They overcome many shortcomings of the traditional "single-signal" fluorescent probes, with fluorescence that can be activated by two analytes of interest order-independently or order-dependently with one output. In this review, we summarize recently published small-molecule double-locked logic gate probes for the optical detection of two bioactive substances in living systems. We envision that this review will attract significant attention from researchers to exploit more powerful functional double-locked logic gate probes.

Small-molecule fluorescent probes for bioactive species in inflammatory disease: arthritis, pneumonia and hepatitis.

Inflammation as an adaptive response underlies a wide variety of physiological and pathological processes. The progression of inflammation is closely intertwined with various bioactive molecules. To dissect the biological mechanisms and physiopathological functions of these molecules, exploitation of versatile detection mean is of great importance. Fluorescence imaging technique has been widely employed to track bioactive species in living systems. As a result, many small-molecule fluorescent probes for bioactive species in inflammatory disease have been developed. However, this interesting and frontier topic hasn't been systematically categorized. Therefore, in this review, we have generalized the construction strategies and biological imaging applications of small-molecule fluorescent probes for various bioactive species, including reactive oxygen/nitrogen/sulfur species, enzyme, mainly in arthritis, pneumonia and hepatitis. Moreover, the future challenges in constructing novel fluorescent probes for inflammatory disease are also present. This review will facilitate the comprehension of superior fluorescent probes for active molecules associated with inflammation.

Universal probe-based SNP genotyping with visual readout: a robust and versatile method.

Detection of single nucleotide polymorphisms (SNPs) is critical for personalized clinical diagnosis, treatment, and medication. Current clinical detection methods suffer from primer dimerization and require the redesigning of reaction systems for different targets, resulting in a time-consuming and laborious process. Here, we present a robust and versatile method for SNP typing by using tailed primers and universal small molecule probes in combination with a visualized lateral flow assay (LFA). This approach enables not only rapid typing of different targets, but also eliminates the interference of primer dimers and enhances the accuracy and reliability of the results. Our proposed universal assay has been successfully applied to the typing of four SNP loci of clinical samples to verify the accuracy and universality, and the results are consistent with those obtained by Sanger sequencing. Therefore, our study establishes a new universal "typing formula" using nucleic acid tags and small molecule probes that provides a powerful genotyping platform for genetic analysis and molecular diagnostics.

Small-molecule fluorogenic probes for mitochondrial nanoscale imaging.

Mitochondria are inextricably linked to the development of diseases and cell metabolism disorders. Super-resolution imaging (SRI) is crucial in enhancing our understanding of mitochondrial ultrafine structures and functions. In addition to high-precision instruments, super-resolution microscopy relies heavily on fluorescent materials with unique photophysical properties. Small-molecule fluorogenic probes (SMFPs) have excellent properties that make them ideal for mitochondrial SRI. This paper summarizes recent advances in the field of SMFPs, with a focus on the chemical and spectroscopic properties required for mitochondrial SRI. Finally, we discuss future challenges in this field, including the design principles of SMFPs and nanoscopic techniques.

Small-molecule probes from bench to bedside: advancing molecular analysis of drug-target interactions toward precision medicine.

Over the past decade, remarkable advances have been witnessed in the development of small-molecule probes. These molecular tools have been widely applied for interrogating proteins, pathways and drug-target interactions in preclinical research. While novel structures and designs are commonly explored in probe development, the clinical translation of small-molecule probes remains limited, primarily due to safety and regulatory considerations. Recent synergistic developments - interfacing novel chemical probes with complementary analytical technologies - have introduced and expedited diverse biomedical opportunities to molecularly characterize targeted drug interactions directly in the human body or through accessible clinical specimens (e.g., blood and ascites fluid). These integrated developments thus offer unprecedented opportunities for drug development, disease diagnostics and treatment monitoring. In this review, we discuss recent advances in the structure and design of small-molecule probes with novel functionalities and the integrated development with imaging, proteomics and other emerging technologies. We further highlight recent applications of integrated small-molecule technologies for the molecular analysis of drug-target interactions, including translational applications and emerging opportunities for whole-body imaging, tissue-based measurement and blood-based analysis.

Recent progress in small-molecule fluorescent probes for in situ imaging and detection of enzyme

Recent progress in small-molecule fluorescent probes for in situ imaging and detection of enzyme

A small-molecule fluorogenic probe for the detection of hypochlorite and its application in the bio-imaging of human breast cancer cells.

A certain amount of hypochlorite can help to regulate the body's defense system while excessive hypochlorite has some complex influence on health. Herein, a thiophene-derived biocompatible turn-on fluorescent probe (TPHZ) was synthesized and characterized for the detection of hypochlorite (ClO-). The fluorescence and colorimetric sensing of the probe followed an ICT OFF strategy. The experimental results showed a remarkable turn on fluorescence enhancement from colorless to bright blue after the addition of ClO- within 130 s in a solvent system having 80% water with high selectivity and a low detection limit of 53.8 nM. The sensing mechanism was attributed to ClO- mediated electrophilic addition to the imine bond which was justified by DFT calculations, and ESI-MS and 1H-NMR titration experiments. The probe was used in an application to visualize ClO- in human breast cancer cells which can be helpful for investigating the functions of hypochlorite in living cells. Finally, by virtue of fine photophysical properties, good sensing performance, good water solubility and low limit of detection, the probe TPHZ was successfully applied to TLC test strips, and commercial bleach and water samples.