Small-molecule Probes Research Articles

Current Progress in the Development of Probes for Targeting α-Synuclein Aggregates.

α-Synuclein aggregates abnormally into intracellular inclusions in Parkinson's disease (PD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), and many other neurological disorders, closely connecting with their pathogenesis. The accurate tracking of α-synuclein by targeting probes is of great significance for early diagnosis, disease monitoring, and drug development. However, there have been no promising α-synuclein targeting probes for clinical application reported so far. This overview focuses on various potential α-synuclein targeting probes reported in the past two decades, including small-molecule fluorescent probes and radiolabeled probes. We provide the current status of the development of the small molecular α-synuclein imaging probes, including properties of promising imaging molecules, strategies of processing new probes, limited progress, and growth prospects in this field, expecting to help in the further development of α-synuclein targeting probes.

Fluorescent probe for simultaneous detection of human serum albumin and sulfite: A theoretical analysis

A small molecule probe for simultaneous detection of Human Serum Albumin (HSA) and Sulfite via distinct fluorescent signals was designed recently. This effective tool provided a significant opportunity to understanding the underlying mechanism of synergistic action between sulfite and HSA in disease. The structural and fluorescent characters of this probe molecule were studied under density functional theory in this work. The different stable conformations of probe C23 were found through theoretical method which can explain the negative experimental fluorescent character of the probe itself. The electron excitation analysis indicated the charge transfer process in the restricted probe C23 (binding to the hydrophobic cavity of HSA) and probe CS (C23 reaction with sulfite) when the molecules were under optical excitation. The theoretical results could be helpful for understanding the electronical properties in the probe and providing the insights for designing new functional probe molecules.

Structural basis for substrate binding and catalysis by a self-alkylating ribozyme.

Ribozymes that react with small-molecule probes have important applications in transcriptomics and chemical biology, such as RNA labeling and imaging. Understanding the structural basis for these RNA-modifying reactions will enable the development of better tools for studying RNA. Nevertheless, high-resolution structures and underlying catalytic mechanisms for members of this ribozyme class remain elusive. Here, we focus on a self-alkylating ribozyme that catalyzes nitrogen-carbon bond formation between a specific guanine and a 2,3-disubstituted epoxide substrate and report the crystal structures of a self-alkylating ribozyme, including both alkylated and apo forms, at 1.71-Å and 2.49-Å resolution, respectively. The ribozyme assumes an elongated hairpin-like architecture preorganized to accommodate the epoxide substrate in a hook-shaped conformation. Observed reactivity of substrate analogs together with an inverse, log-linear pH dependence of the reaction rate suggests a requirement for epoxide protonation, possibly assisted by the ether oxygens within the substrate.

Covalent Biosensing Polymer Chain Reaction Enabling Periphery Blood Testing to Predict Tumor Invasiveness with a Platelet Procancerous Protein.

Periphery blood testing is an attractive and relatively less invasive way of early cancer screening. In this work, based on the latest understanding of the pivotal role of platelets in promoting cancer invasion, a method for detecting a procancerous protein overexpressed both on platelets and in cancer cells is developed. As a kinase, the enzymatic activity, abundance, and self-phosphorylation of this protein are all important factors influencing its procancerous activity. To simultaneously determine these three important biochemical parameters, electrochemical control is called upon to connect or disconnect a polymer chain reaction (PCR) primer with a small-molecule synthetic probe, and with the target protein, in a target-specific manner. The resulting PCR signal amplification greatly improves the sensitivity of the design and also enables direct detection of the protein and its catalytic activity as well as its self-phosphorylation in clinical periphery blood samples from hepatocellular carcinoma (HCC) patients. This may point to future application of the proposed method in the early screening of HCC to assist its diagnosis and treatment.

Discovery of small-molecule fluorescent probes for C-Met

C-mesenchymal-epithelia transition factor (c-Met) is highly expressed in various solid tumors such as gastric cancer, liver cancer, and lung cancer, playing a pivotal role in the growth, maintenance, and development of different tumor cells. In this study, three small-molecule fluorescent probes (5, 11, 16) targeting c-Met were developed, and their design strategies were also initially explored. In general, the fluorescence properties of the probes themselves could meet the imaging requirements, and they have shown sufficient inhibitory activities against c-Met, especially probe 16, reflecting the targeting and acceptance. Also, fluorescence polarization assays and flow cytometry analysis verified the binding between the probes and c-Met. Cell imaging confirmed that these probes could be used to label c-Met on living cells. It is of positive significance for the development of c-Met kinase inhibitors and tumor pathology research.

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth.

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

Progress in small-molecule fluorescent probes for nucleic acids

<p indent="0mm">Nucleic acids (NAs), including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are biological macromolecular substances formed through the polymerization of nucleotide monomers. NAs contribute to the growth, development, and reproduction of living organisms. Due to developments in fluorescence-imaging technology, organic small-molecule fluorescent probes have become robust tools for imaging nucleic acid in cells, tissues, and <italic>in vivo</italic>. In this paper, based on the fluorophores, we systematically summarized the recent (2000–2021) progress in the development of organic small-molecule fluorescent probes for detecting NAs. In addition, the design strategies, reaction mechanisms, and biological applications of these fluorescent chemosensors were discussed in this review. Finally, the challenges and prospects of this promising field were discussed.

Recent advance in dual-functional luminescent probes for reactive species and common biological ions.

The chemical microenvironment in cells is extremely complex involving numerous species with drastically different concentration as well as temporal and spatial distribution. Especially, reactive species including ROS, RNS, RSS, and many inorganic ions are recognized to play very important roles. The concentrations of these species are constantly regulated and often depend on each other. While fluorescent probes have been widely used to study various cell biological processes, those simultaneously probing two species just emerged recently. In this review, we highlight dual-functional luminescent probes for the detection and fluorescence imaging of two and more analytes in cells. The content will cover small-molecule synthetic fluorescent probes, DNA nanoassemblies, and nanoparticle-based nanoprobes. The target analytes include reactive species such as ROS, RNS, and RSS and other ions and molecules such as H+, Cl-, Ca2+, Cu2+, and O2.

Small-molecule fluorescent probes: big future for specific bacterial labeling and infection detection.

Bacterial infections remain a global healthcare problem that is particularly attributed to the spread of antibiotic resistance and the evolving pathogenicity. Accurate and swift approaches for infection diagnosis are urgently needed to facilitate antibiotic stewardship and effective medical treatment. Direct optical imaging for specific bacterial labeling and infection detection offers an attractive prospect of precisely monitoring the infectious disease status and therapeutic response in real time. This feature article focuses on the recent advances of small-molecule probes developed for fluorescent imaging of bacteria and infection, which covers the probe design, responsive mechanisms and representative applications. In addition, the perspective and challenges to advance small-molecule fluorescent probes in the field of rapid drug-resistant bacterial detection and clinical diagnosis of bacterial infections are discussed. We envision that the continuous advancement and clinical translations of such a technique will have a strong impact on future anti-infective medicine.

Small-molecule fluorescence-based probes for aging diagnosis

Aging is a time-dependent decline in physiological function that affects most organisms and is the major risk factor for many non-communicable diseases. The early diagnosis of aging is critical for the treatment of aging and aging-related diseases. In recent years, extensive efforts have attempted to accurately diagnose aging. To date, multiple types of fluorescent probes for various age-related biomarkers have been developed, with the aims of achieving rapid, precise diagnosis of the aging process and evaluating the efficacy of anti-aging drugs. This review summarizes recent research progress in small-molecule fluorescence-based probes for aging diagnosis, and further discusses the challenges and opportunities in this field.

Inhibition of SC4MOL and HSD17B7 shifts cellular sterol composition and promotes oligodendrocyte formation.

While the cholesterol biosynthesis pathway has been extensively studied, recent work has forged new links between inhibition of specific sterol pathway enzymes, accumulation of their unique sterol substrates, and biological areas as diverse as cancer, immunology, and neurodegenerative disease. We recently reported that dozens of small molecules enhance formation of oligodendrocytes, a glial cell type lost in multiple sclerosis, by inhibiting CYP51, Sterol 14-reductase, or EBP and inducing cellular accumulation of their 8,9-unsaturated sterol substrates. Several adjacent pathway enzymes also have 8,9-unsaturated sterol substrates but have not yet been evaluated as potential targets for oligodendrocyte formation or in many other biological contexts, in part due to a lack of available small-molecule probes. Here, we show that genetic suppression of SC4MOL or HSD17B7 increases the formation of oligodendrocytes. Additionally, we have identified and optimized multiple potent new series of SC4MOL and HSD17B7 inhibitors and shown that these small molecules enhance oligodendrocyte formation. SC4MOL inhibitor CW4142 induced accumulation of SC4MOL's sterol substrates in mouse brain and represents an in vivo probe of SC4MOL activity. Mechanistically, the cellular accumulation of these 8,9-unsaturated sterols represents a central driver of enhanced oligodendrocyte formation, as exogenous addition of purified SC4MOL and HSD17B7 substrates but not their 8,9-saturated analogs promotes OPC differentiation. Our work validates SC4MOL and HSD17B7 as novel targets for promoting oligodendrocyte formation, underlines a broad role for 8,9-unsaturated sterols as enhancers of oligodendrocyte formation, and establishes the first high-quality small molecules targeting SC4MOL and HSD17B7 as novel tools for probing diverse areas of biology.

Triphenylamine-based small-molecule fluorescent probes.

Ammonia with the three hydrogens substituted by phenyls is known as triphenylamine (TPA), and is one of the most useful compounds because of its vast practical applications. Chemists have produced thousands of TPA derivatives to date. Because of its biocompatibility and structural features, it has been widely used in the fields of molecular recognition, molecular imaging, materials chemistry, and also in biology and medical science. Its strong electron-donating ability encourages scientists to produce different types of probes for molecular recognition. This review is based on recent developments and advances in TPA-based small molecular fluorescent probes within the time period 2010-2021. This extensive review may expedite improvements in more advanced fluorescent probes for vast and stimulating applications in the future.

Recent advances in small-molecule fluorescent probes for studying ferroptosis.

Ferroptosis is an iron-dependent, non-apoptotic form of programmed cell death driven by excessive lipid peroxidation (LPO). Mounting evidence suggests that the unique modality of cell death is involved in the development and progression of several diseases including cancer, cardiovascular diseases (CVDs), neurodegenerative disorders, etc. However, the pathogenesis and signalling pathways of ferroptosis are not fully understood, possibly due to the lack of robust tools for the highly selective and sensitive imaging of ferroptosis analytes in complex living systems. Up to now, various small-molecule fluorescent probes have been applied as promising chemosensors for studying ferroptosis through tracking the biomolecules or microenvironment-related parameters in vitro and in vivo. In this review, we comprehensively reviewed the recent development of small-molecule fluorescent probes for studying ferroptosis, with a focus on the analytes, design strategies and bioimaging applications. We also provided new insights to overcome the major challenges in this emerging field.

Small-molecule fluorescent probes based on covalent assembly strategy for chemoselective bioimaging.

Fluorescent probes have been widely studied and applied in environment and health analysis, where among them small molecular “covalent assembly” probes are a novel type of reaction probes with many advantages, including no background interference, remarkable colorimetric change, rapid response, high sensitivity, and strong fluorescent signal. During the past decade, significant contributions have been made globally to both the application and mechanism of covalent assembly probes. In this review, we summarize the recent development of covalent assembly probes, classifying them based on different analytes, such as anions, metal ions, small biological molecules, reactive oxidative spices (ROS), reactive nitrogen species (RNS), nerve agent mimics, and enzymes, and introduce their detection mechanism in detail. Furthermore, the perspective on the next generation of covalent-assembly probes toward biomolecules imaging is presented.

Resolution and characterization of confinement- and temperature-dependent dynamics in solvent phases that surround proteins in frozen aqueous solution by using spin-probe EPR spectroscopy.

Spin probe electron paramagnetic resonance spectroscopy is applied to characterize the dynamics of concentric hydration and mesophase solvent domains that surround proteins within the ice boundary in frozen aqueous solutions. The solvent dynamics are tuned by variation of temperature (190-265K) and by the degree of ice boundary confinement, which is modulated by the volume of added cryosolvent (0-~50Å separation distance from protein surface). Goals are to: (1) characterize the protein-coupled solvent dynamics on correlation time scales of ~10-10<τ<10-7s, and spatial scales from protein surface to periphery of the surrounding solution, from the perspective of a free, small-molecule (~7Å diameter) probe, and (2) reveal properties of the solvent-protein coupling that can be correlated with protein functions, that are measureable under the same conditions. Rotational mobility of the nitroxide spin probe, TEMPOL, resolves and tracks two solvent components, the protein-associated domain (PAD; akin to hydration layer) and surrounding mesodomain, through their distinct temperature- and confinement-dependent values of τ and normalized weight. Detailed protocols are described for simulation of two-component nitroxide EPR spectra, which are categorized by line shape regime and guided by a library of template spectra and simulation parameters derived from two model soluble globular proteins. The order-disorder transition in the PAD, which is a universal feature of protein-coupled solvent dynamics, provides a well-defined, tunable property for elucidating mechanism in solvent-protein-function dynamical coupling. The low-temperature mesodomain system and EPR spin probe method are generally applicable to reveal solvent contributions to a broad range of macromolecule-mediated biological processes.

Recent progress in small-molecule fluorescent probes for endoplasmic reticulum imaging in biological systems.

Endoplasmic reticulum (ER) is an indispensable organelle in eukaryotic cells involved in protein synthesis and processing, as well as calcium storage and release. Therefore, maintaining the quality of ER is of great importance for cellular homeostasis. Aberrant fluctuations of bioactive species in the ER will result in homeostasis disequilibrium and further cause ER stress, which has evolved to contribute to the pathogenesis of various diseases. Therefore, the real-time monitoring of various bioactive species in the ER is of high priority to ascertain the mysterious roles of ER, which will contribute to unveiling the corresponding mechanism of organism disturbances. Recently, fluorescence imaging has emerged as a robust technique for the direct visualization of molecular events due to its outstanding sensitivity, high temporal-spatial resolution and noninvasive nature. In this review, we comprehensively summarize the recent progress in design strategies, bioimaging applications, potential directions and challenges of ER-targetable small-molecular fluorescent probes.

Autophagy takes it all - autophagy inducers target immune aging.

ABSTRACTAutophagy, as the key nutrient recycling pathway, enables eukaryotic cells to adapt to surging cellular stress during aging and, thereby, delays age-associated deterioration. Autophagic flux declines with age and, in turn, decreases in autophagy contribute to the aging process itself and promote senescence. Here, we outline how autophagy regulates immune aging and discuss autophagy-inducing interventions that target senescent immune cells, which are major drivers of systemic aging. We examine how cutting-edge technologies, such as single-cell omics methods hold the promise to capture the complexity of molecular and cellular phenotypes associated with aging, driving the development of suitable putative biomarkers and clinical bioassays. Finally, we debate the urgency to initiate large-scale human clinical trials. We give special preference to small molecule probes and to dietary interventions that can extend healthy lifespan and are affordable for most of the world's population.

A ligand selection strategy to customize small molecule probes for activity-based protein profiling (LS-ABPP).

Activity-based probes (ABPs) are the key components of activity-based protein profiling (ABPP). However, designing a probe that shows target-specific as well as site-selective binding can be a challenging and time-consuming task, often requiring complex synthetic procedures to provide a selection of probes from which to choose the ideal one. In this chapter, we present a ligand selection (LS) approach that allows us to rapidly diversify probe molecules in order to meet the steric and electronic demands of the binding site of any target enzyme. The central element of this method is a trifunctional LS probe synthesized from tyrosine in five steps, consisting of a highly reactive pentafluorophenyl (PFP) ester in addition to an electrophilic chloroacetamide warhead, and a bioorthogonal alkyne reporter group. By reacting a variety of primary amine ligands with the PFP ester, a probe library is created and screened for optimal binding characteristics to the target enzyme. With the optimized probe in hand, a compound library is subsequently screened by competitive profiling to identify potential enzyme inhibitors. Conveniently, this protocol is highly adaptable to a large variety of target proteins, representing a valuable tool for enzyme characterization and the discovery of enzyme inhibitors. Here, we apply this method exemplarily to the cysteine protease 3CLpro of the coronavirus SARS-CoV-2.

Metalloenzymes involved in carotenoid biosynthesis in plants.

Carotenoids are a family of pigment compounds, a subset of which are precursors for vitamin A biosynthesis. These pigments are derived from isopentenyl pyrophosphate (IPP), with geranylgeranyl diphosphate being the first metabolite unique to carotenoid biosynthesis in plants, algae, fungi, some bacteria, and arthropods. This chapter highlights the metal-dependent enzymes involved in synthesizing carotenoids in plants and the current state of knowledge of their cofactors and mechanisms. Emphasis is given to spectroscopic methods used to characterize metal centers. The recently discovered heme-dependent isomerase Z-ISO is presented as a case study in how to interrogate a metalloenzyme. Use of UV-vis, electron paramagnetic resonance, and magnetic circular dichroism spectroscopies of a metal center at various oxidation states and with external small molecule probes (CN-, CO, and NO) can provide information about the nature of the metal center, the identity of its ligands, and its mechanism of action. Z-ISO is a histidine/cysteine ligated heme-dependent enzyme that is only active in the ferrous state and possesses redox-linked ligand switching. The choice and design of experiments are discussed as well as the conclusions that can be drawn.

Selective and swift-responsive “off-on” rhodamine B based chemosensors: Recognition of multi-metal ions, on-site sensing of Fe(III) in water samples and bioimaging in aqueous media

Distinct “off–on” rhodamine B based chemosensors 3',6'-bis(diethylamino)-2-(2-(diethylamino)ethyl) spiro[isoindoline-1,9′-xanthen]-3-one (2a); 3′,6′-bis(diethylamino)-2-(2-(dimethylamino)ethyl) spiro[isoindole-1,9′-xanthen]- 3-one (2b) are synthesized in a facile single-step reaction and characterized by X-ray crystallography, NMR, Mass, and FT-IR techniques. 2a, 2b; selectively detected Fe+3, Sn+2, and Cr+3, in aqueous media over 17 other environmentally relevant cations. Furthermore, 2a, 2b showed high selectivity towards Fe+3 ion with ∼ 62 fold (2a), ∼21 fold (2b) enhanced emission intensity. A prominent pink color, in < 1 min (∼40 s) was detected under naked eye on sensing Fe+3. Enhanced emission was rationalized by natural transition orbital analysis using DFT and TD-DFT methods. Detection limit and binding constant of Fe+3 for 2a and 2b are estimated as 51.25 ppb (73 nM), 40.3 ppb (59.7 nM), and 0.80 × 103 M−1, 4.59 × 103 M−1, each respectively, indicating their efficiency towards Fe+3 sensing. A test strip method was developed for the on-site qualitative detection of target metal ions, and practical applicability was demonstrated by determining the concentration of Fe+3 ion in real water samples. Probes 2a, 2b further tested in human gastric adenocarcinoma (AGS) cell-lines via in-vitro imaging. The increased emission intensity in 2a, 2b on binding to Fe+3 in AGS cells indicate their potentiality as small-molecule based biological fluorescent probes.