Current and Future of "Turn-On" Based Small-Molecule Copper Probes for Cuproptosis.

Increasing evidence shows that abnormal copper (Cu) metabolism is highly related to many diseases, such as Alzheimer's disease, Wilson's disease, hematological malignancies and Menkes disease. Very recently, cuproptosis, a Cu-dependent, programmed cell death was firstly described by Tsvetkov et al. in 2022. Their findings may provide a new perspective for the treatment of related diseases. However, the concrete mechanisms of these diseases, especially cuproptosis, remain completely unclear, the reason of which may be a lack of reliable tools to conduct highly selective, sensitive and high-resolution imaging of Cu in complex life systems. So far, numerous small-molecular fluorescent probes have been designed and utilized to explore the Cu signal pathway. Among them, fluorescence turn-on probes greatly enhance the resolution and accuracy of imaging and may be a promising tool for research of investigation into cuproptosis. This review summarizes the probes developed in the past decade which have the potential to study cuproptosis, focusing on the design strategies, luminescence mechanism and biological-imaging applications. Besides, we put forward some ideas concerning the design of next-generation probes for cuproptosis, aiming to tackle the main problems in this new field. Furthermore, the prospect of cuproptosis in the treatment of corresponding diseases is also highlighted.

Deuteration Degree-Controllable Methylation via a Cascade Assembly Strategy using Methylamine-Water as Methyl Source.

We present a novel and effective photocatalytic method for the methylation of ß-diketones with controllable degrees of deuterium incorporation via development of new methyl sources. By utilizing a methylamine-water system as the methyl precursor and a cascade assembly strategy for deuteration degree control, we synthesized methylated compounds with varying degrees of deuterium incorporation, showcasing the versatility of this approach. We examined a range of ß-diketone substrates and synthesized key intermediates for drug and bioactive compounds with varying degrees of deuterium incorporation, ranging from 0 to 3. We also investigated and discussed the postulated reaction pathway. This work demonstrates the utility of readily available reagents, methylamines and water, as a new methyl source, and provides a simple and efficient strategy for the synthesis of degree-controllable deuterium-labelled compounds.

Liposomes as Multifunctional Nano-Carriers for Medicinal Natural Products.

Although medicinal natural products and their derivatives have shown promising effects in disease therapies, they usually suffer the drawbacks in low solubility and stability in the physiological environment, low delivery efficiency, side effects due to multi-targeting, and low site-specific distribution in the lesion. In this review, targeted delivery was well-guided by liposomal formulation in the aspects of preparation of functional liposomes, liposomal medicinal natural products, combined therapies, and image-guided therapy. This review is believed to provide useful guidance to enhance the targeted therapy of medicinal natural products and their derivatives.

Multi-Functional Liposome: A Powerful Theranostic Nano-Platform Enhancing Photodynamic Therapy.

Although photodynamic therapy (PDT) has promising advantages in almost non-invasion, low drug resistance, and low dark toxicity, it still suffers from limitations in the lipophilic nature of most photosensitizers (PSs), short half-life of PS in plasma, poor tissue penetration, and low tumor specificity. To overcome these limitations and enhance PDT, liposomes, as excellent multi-functional nano-carriers for drug delivery, have been extensively studied in multi-functional theranostics, including liposomal PS, targeted drug delivery, controllable drug release, image-guided therapy, and combined therapy. This review provides researchers with a useful reference in liposome-based drug delivery.

Visible light-induced mono-bromination of arenes with BrCCl3.

A highly efficient and regioselective bromination of electron-rich arenes and heteroarenes using commercially available BrCCl3 as a "Br" source has been developed. The reaction was performed in air under mild conditions with photocatalyst Ru(bpy)3Cl2·6H2O, avoiding the usage of strong acids and strong oxidants. Mono-brominated products were obtained with medium to excellent yields (up to 94%). This strategy has shown good compatibility and high para-selectivity, which will facilitate the complicated synthesis.

Fluorogenic Probes/Inhibitors of ß-Lactamase and their Applications in Drug-Resistant Bacteria.

ß-Lactam antibiotics are generally perceived as one of the greatest inventions of the 20th century, and these small molecular compounds have saved millions of lives. However, upon clinical application of antibiotics, the ß-lactamase secreted by pathogenic bacteria can lead to the gradual development of drug resistance. ß-Lactamase is a hydrolase that can efficiently hydrolyze and destroy ß-lactam antibiotics. It develops and spreads rapidly in pathogens, and the drug-resistant bacteria pose a severe threat to human health and development. As a result, detecting and inhibiting the activities of ß-lactamase are of great value for the rational use of antibiotics and the treatment of infectious diseases. At present, many specific detection methods and inhibitors of ß-lactamase have been developed and applied in clinical practice. In this Minireview, we describe the resistance mechanism of bacteria producing ß-lactamase and further summarize the fluorogenic probes, inhibitors of ß-lactamase, and their applications in the treatment of infectious diseases. It may be valuable to design fluorogenic probes with improved selectivity, sensitivity, and effectiveness to further identify the inhibitors for ß-lactamases and eventually overcome bacterial resistance.

Multifunctional Liposome: A Bright AIEgen-Lipid Conjugate with Strong Photosensitization.

Liposomes have been used as popular drug delivery systems for cancer therapy. However, it is difficult to track traditional liposome delivery systems in an efficient and stable fashion to assess their delivery efficacy and biodistribution after administration. Meanwhile, conventional fluorescent liposomes containing optical tracers face the challenge of aggregation-caused quenching. Herein, we report a strategy for the integration of an aggregation-induced emission fluorogen with a liposome to yield an AIEgen-lipid conjugate, termed "AIEsome". The AIEsome exhibits bright red fluorescence along with great photostability and biocompatibility, and can be used for in vitro cancer cell labeling and in vivo tumor targeting. Meanwhile, benefiting from the excellent photosensitizing ability of the AIEgen and its good oxygen exposure in aqueous media, the AIEsome also performs well in efficient photodynamic therapy (PDT) for both in vitro cancer cell ablation and in vivo antitumor therapy after white light illumination.

A Highly Efficient and Photostable Photosensitizer with Near-Infrared Aggregation-Induced Emission for Image-Guided Photodynamic Anticancer Therapy.

Photodynamic therapy (PDT), which relies on photosensitizers (PS) and light to generate reactive oxygen species to kill cancer cells or bacteria, has attracted much attention in recent years. PSs with both bright emission and efficient singlet oxygen generation have also been used for image-guided PDT. However, simultaneously achieving effective 1 O2 generation, long wavelength absorption, and stable near-infrared (NIR) emission with low dark toxicity in a single PS remains challenging. In addition, it is well known that when traditional PSs are made into nanoparticles, they encounter quenched fluorescence and reduced 1 O2 production. In this contribution, these challenging issues have been successfully addressed through designing the first photostable photosensitizer with aggregation-induced NIR emission and very effective 1 O2 generation in aggregate state. The yielded nanoparticles show very effective 1 O2 generation, bright NIR fluorescence centered at 820 nm, excellent photostability, good biocompatibility, and negligible dark in vivo toxicity. Both in vitro and in vivo experiments prove that the nanoparticles are excellent candidates for image-guided photodynamic anticancer therapy.

Rational Design of a Red-Emissive Fluorophore with AIE and ESIPT Characteristics and Its Application in Light-Up Sensing of Esterase.

The development of red fluorophores with efficient solid-state emission is still challenging. Herein, a red fluorophore 1 with aggregation-induced emission (AIE) and excited-state intramolecular proton transfer (ESIPT) characteristics is rationally designed and facilely synthesized by attaching an electron-donor diethylamine and an electron-acceptor maleonitrile group to salicyladazine. In contrast to many red fluorophores which undergo serious aggregation-caused quenching (ACQ), compound 1 emits bright red fluorescence (λem = 650 nm, ΦF = 24.3%) in the solid state with a large Stokes shift of 174 nm. Interestingly, control compounds 2 and 3, which have similar structures as 1, exhibit obvious aggregation-caused quenching (ACQ) characteristics. The difference in the crystal structures of 1, 2, and 3 reveals that the interplanar spacing among molecules plays a decisive role in realizing the AIE characteristics of 1. Moreover, when the hydroxyl group of 1 was substituted by an esterase reactive acetoxyl, a fluorescence light-up probe 4 was developed for sensing of esterase based on the selective reaction between 4 and esterase to generate the AIE and ESIPT active molecule 1. The linear range for in vitro quantification of esterase is 0.01-0.15 U/mL with a detection limit of 0.005 U/mL. Probe 4 was also successfully applied to image esterase in mitochondria of living cells.

A reusable and naked-eye molecular probe with aggregation-induced emission (AIE) characteristics for hydrazine detection.

We report a fluorogenic probe for naked-eye sensing of hydrazine in solution and in the gaseous phase. The probe based on tetraphenylethylene (TPE) with aggregation-induced emission (AIE) characteristics shows OFF-ON fluorescence as observed by thin-layer chromatography (TLC) upon treatment with hydrazine. Specifically, the fluorescence of the probe was quenched due to the attached N[double bond, length as m-dash]N group, which can be reduced to -NH-NH- in the presence of hydrazine to turn on the fluorescence. The reduced intermediate can be easily oxidized in air to regenerate the original probe for recyclable usage. Both fluorometric and colorimetric readings were achieved by TLC with high sensitivity and excellent selectivity. This study thus represents a simple example of a reusable and naked-eye molecular probe for monitoring environmental hazards. Finally, the probe has also been applied to detect hydrazine in live cells.

A Porphyrin-Based Conjugated Polymer for Highly Efficient In Vitro and In Vivo Photothermal Therapy.

Conjugated polymers have been increasingly studied for photothermal therapy (PTT) because of their merits including large absorption coefficient, facile tuning of exciton energy dissipation through nonradiative decay, and good therapeutic efficacy. The high photothermal conversion efficiency (PCE) is the key to realize efficient PTT. Herein, a donor-acceptor (D-A) structured porphyrin-containing conjugated polymer (PorCP) is reported for efficient PTT in vitro and in vivo. The D-A structure introduces intramolecular charge transfer along the backbone, resulting in redshifted Q band, broadened absorption, and increased extinction coefficient as compared to the state-of-art porphyrin-based photothermal reagent. Through nanoencapsulation, the dense packing of a large number of PorCP molecules in a single nanoparticle (NP) leads to favorable nonradiative decay, good photostability, and high extinction coefficient of 4.23 × 104 m-1 cm-1 at 800 nm based on porphyrin molar concentration and the highest PCE of 63.8% among conjugated polymer NPs. With the aid of coloaded fluorescent conjugated polymer, the cellular uptake and distribution of the PorCP in vitro can be clearly visualized, which also shows effective photothermal tumor ablation in vitro and in vivo. This research indicates a new design route of conjugated polymer-based photothermal therapeutic materials for potential personalized theranostic nanomedicine.

Real-Time Specific Light-Up Sensing of Transferrin Receptor: Image-Guided Photodynamic Ablation of Cancer Cells through Controlled Cytomembrane Disintegration.

Transferrin receptor (TfR) represents a unique target for specific imaging of cancer cells and targeted delivery of therapeutic reagents. Detection and qualification of TfR is thus of great importance for cancer diagnosis and therapy. In this contribution, a light-up probe TPETH-2T7 was developed by conjugating a red-emissive photosensitizer with aggregation-induced emission (AIE) characteristics to a TfR-targeting peptide T7. The probe is almost nonemissive by itself, but it gives turn-on fluorescence in the presence of TfR with a detection limit of 0.45 µg/mL. Cellular experiments show that the probe specifically binds to TfR-overexpressed cancer cells. Real-time imaging results reveal that the probe stains the MDA-MB-231 cell membrane in 30 min, which is followed by probe internalization. Experiments on image-guided photodynamic cancer ablation show that the therapeutic performance is better when TPETH-2T7 is localized on the cell membrane as compared to that being internalized into cells. Confocal laser scanning microscopy (CLSM) study reveals that cytomembrane disintegration allows quick ablation of MDA-MB-231 cells.

Bioorthogonal Turn-On Probe Based on Aggregation-Induced Emission Characteristics for Cancer Cell Imaging and Ablation.

Bioorthogonal turn-on probes have been widely utilized in visualizing various biological processes. Most of the currently available bioorthogonal turn-on probes are blue or green emissive fluorophores with azide or tetrazine as functional groups. Herein, we present an alternative strategy of designing bioorthogonal turn-on probes based on red-emissive fluorogens with aggregation-induced emission characteristics (AIEgens). The probe is water soluble and non-fluorescent due to the dissipation of energy through free molecular motion of the AIEgen, but the fluorescence is immediately turned on upon click reaction with azide-functionalized glycans on cancer cell surface. The fluorescence turn-on is ascribed to the restriction of molecular motion of AIEgen, which populates the radiative decay channel. Moreover, the AIEgen can generate reactive oxygen species (ROS) upon visible light (λ=400-700 nm) irradiation, demonstrating its dual role as an imaging and phototherapeutic agent.

A FRET probe with AIEgen as the energy quencher: dual signal turn-on for self-validated caspase detection.

The accurate detection of biological substances is highly desirable to study various biological processes and evaluate disease progression. Herein, we report a self-validated fluorescent probe which is composed of a coumarin fluorophore as the energy donor and a fluorogen with aggregation-induced emission characteristics (AIEgen) as the energy quencher linked through a caspase-3 specific peptide substrate. Unlike the traditionally widely studied fluorescence resonance energy transfer (FRET) probes, our new generation of FRET probe is non-fluorescent itself due to the energy transfer as well as the dissipation of the acceptor energy through the free molecular motion of AIEgen. Upon interaction with caspase-3, the probe displays strong green and red fluorescent signals synchronously due to the separation of the donor-quencher and aggregation of the released AIEgen. The fluorescence turn-on with dual signal amplification allows real-time and self-validated enzyme detection with a high signal-to-background ratio, providing a good opportunity to accurately monitor various biological processes in a real-time manner.

In situ proteome profiling of C75, a covalent bioactive compound with potential anticancer activities.

A library of cell-permeable, minimally tagged C75 analogues was synthesized and used to uncover biological targets in human liver cancer cells. Known targets of C75, namely FASN and CPT1A, together with other unknown targets, including PDIA3, TFRC, and GAPDH, were thus identified.

A tuned affinity-based staurosporine probe for in situ profiling of protein kinases.

A clickable and cell-permeable affinity-based probe (AfBP) was designed from staurosporine, by incorporating an electrophilic chloroacetamide warhead to facilitate in situ proteome labeling of a range of potential kinase targets.

Targeting lipid esterases in mycobacteria grown under different physiological conditions using activity-based profiling with tetrahydrolipstatin (THL).

Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Guérin cultured under replicating and non-replicating conditions. THL targets α/ß-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes.

Cell-based proteome profiling using an affinity-based probe (AfBP) derived from 3-deazaneplanocin A (DzNep).

3-Deazaneplanocin A (DzNep), a global histone methylation inhibitor, has attracted significant interest in epigenetic research in recent years. The molecular mechanism of action and the cellular off-targets of DzNep, however, are still not well-understood. Our aim was to develop novel DzNep-derived small-molecule probes suitable to be used in live mammalian cells for identification of potential cellular targets of DzNep under physiologically relevant settings. In the current study, we have successfully designed, synthesized, and tested one such probe, called DZ-1. DZ-1 is a 'clickable' affinity-based probe (AfBP) derived from DzNep with minimal structural modifications. The probe was found to be highly cell-permeable, and possessed similar anti-apoptotic activities as DzNep in MCF-7 mammalian cells. Two additional control probes were made as negative labeling/pull-down probes in order to minimize false identification of background proteins due to unavoidable, intrinsic nonspecific photo-crosslinking reactions. All three probes were subsequently used for in-situ proteome profiling in live mammalian cells, followed by large-scale pull-down/LC-MS/MS analysis for identification of potential cellular protein targets that might interact with DzNep in native cellular environments. Our LC-MS/MS results revealed some highly enriched proteins that had not been reported as potential DzNep targets. These proteins might constitute unknown cellular off-targets of DzNep. Though further validation experiments are needed in order to unequivocally confirm these off-targets, our findings shed new light on the future use of DzNep as a validated chemical probe for epigenetic research and as a potential drug candidate for cancer therapy.

Ugi reaction-assisted rapid assembly of affinity-based probes against potential protein tyrosine phosphatases.

The multi-component Ugi reaction has been employed to assemble a small library of affinity-based probes (AfBPs) that target potential protein tyrosine phosphatases. The probes showed good labelling of PTP1B and MptpB, and were subsequently used to label endogenous PTP1B in MCF-7 cell lysates.