A Radical-Generating Probe to Release Free Fluorophores and Identify Artemisinin-Sensitive Cancer Cells

The smart light-up probes have been extensively developed to image various enzymes and other bioactive molecules. Upon activation, these probes result in light-up fluorophores that exist in a protein-bound or a free form. The difference between these two forms has not yet been reported. Here, we present a pair of smart light-up probes that generate a protein-bound fluorophore and a free fluorophore upon activation by heme. Probe 8 generated a radical-attached fluorophore that predominantly existed in the free form, while probe 10 generated an α,β-unsaturated ketone-attached fluorophore that showed extensive labeling of proteins. In live-cell imaging, probe 8 showed greater fluorescence intensity than probe 10 when low concentrations (0.1–5 μM) of the probes were used, but probe 8 was less fluorescent than probe 10 when the concentrations of the probes were high (10 μM). Finally, probe 8 was used to reflect the activation level of the endoperoxide bond in cancer cells and to effectively distinguish ART-sensitive cancer cells from ART-insensitive ones

Zinc(II)-Tetradentate-Coordinated Probe with Aggregation-Induced Emission Characteristics for Selective Imaging and Photoinactivation of Bacteria

The emergence of drug-resistant bacterial pathogens highlights an urgent need for new therapeutic options. Photodynamic therapy (PDT) has emerged as a potential alternative to antibiotics to kill bacteria, which has been used in clinical settings. PDT employs photosensitizers (PSs), light, and oxygen to kill bacteria by generating highly reactive oxygen species (ROS). PDT can target both external and internal structures of bacteria, which does not really require the PSs to enter bacteria. Therefore, bacteria can hardly develop resistance to PDT. However, most of the PSs reported so far are hydrophobic and tend to form aggregates when they interact with bacteria. The aggregation could cause fluorescence quenching and reduce ROS generation, which generally compromises the effects of both imaging and therapy. In this contribution, we report on a Zn­(II)-tetradentate-coordinated red-emissive probe with aggregation-induced emission characterization. The probe could selectively image bacteria over mammalian cells. Moreover, the probe shows potent phototoxicity to both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Bacillus subtilis)

Artemisinin and AIEgen Conjugate for Mitochondria-Targeted and Image-Guided Chemo- and Photodynamic Cancer Cell Ablation

Cell organelle targeting is a promising approach for cancer therapy. We herein report a light-up probe (tetraphenylethenethiophene (TPETH)-Mito-1ART) to co-deliver artemisinin (ART) and an aggregation-induced emission (AIE) photosensitizer to cancer cell mitochondria for image-guided combination cancer cell ablation. This probe contains a TPETH core, two mitochondria targeting arms with ART on one arm, which show high specificity toward cancer cells over normal ones, predominant accumulation, and fluorescence turn-on in mitochondria. The fresh heme produced in mitochondria quickly activates ART, and the direct generation of reactive oxygen species at mitochondria promotes photodynamic therapy (PDT) performance. The incorporation of ART and PDT leads to a largely improved cancer cell ablation efficacy with a synergistic effect, which could quickly depolarize mitochondrial membrane and largely reduce cancer migration activity. This co-delivery strategy provides great potentials for subcellular organelle-targeted and image-guided combination cancer cell ablation

Cell-Based Proteome Profiling of Potential Dasatinib Targets by Use of Affinity-Based Probes

Protein kinases (PKs) play an important role in the development and progression of cancer by regulating cell growth, survival, invasion, metastasis, and angiogenesis. Dasatinib (BMS-354825), a dual Src/Abl inhibitor, is a promising therapeutic agent with oral bioavailability. It has been used for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). Most kinase inhibitors, including Dasatinib, inhibit multiple cellular targets and do not possess exquisite cellular specificity. Recent efforts in kinase research thus focus on the development of large-scale, proteome-wide chemical profiling methods capable of rapid identification of potential cellular (on- and off-) targets of kinase inhibitors. Most existing approaches, however, are still problematic and in many cases not compatible with live-cell studies. In this work, we have successfully developed a cell-permeable kinase probe (<b>DA-2</b>) capable of proteome-wide profiling of potential cellular targets of Dasatinib. In this way, highly regulated, compartmentalized kinase–drug interactions were maintained. By comparing results obtained from different proteomic setups (live cells, cell lysates, and immobilized affinity matrix), we found <b>DA-2</b> was able to identify significantly more putative kinase targets. In addition to Abl and Src family tyrosine kinases, a number of previously unknown Dasatinib targets have been identified, including several serine/threonine kinases (PCTK3, STK25, eIF-2A, PIM-3, PKA C-α, and PKN2). They were further validated by pull-down/immunoblotting experiments as well as kinase inhibition assays. Further studies are needed to better understand the exact relevance of Dasatinib and its pharmacological effects in relation to these newly identified cellular targets. The approach developed herein should be amenable to the study of many of the existing reversible drugs/drug candidates

Concentration-Dependent Enrichment Identifies Primary Protein Targets of Multitarget Bioactive Molecules

Multitarget bioactive molecules (MBMs) are of increasing importance in drug discovery as they could produce high efficacy and a low chance of resistance. Several advanced approaches of quantitative proteomics were developed to accurately identify the protein targets of MBMs, but little study has been carried out in a sequential manner to identify primary protein targets (PPTs) of MBMs. This set of proteins will first interact with MBMs in the temporal order and play an important role in the mode of action of MBMs, especially when MBMs are at low concentrations. Herein, we describe a valuable observation that the result of the enrichment process is highly dependent on concentrations of the probe and the proteome. Interestingly, high concentrations of probe and low concentrations of incubated proteome will readily miss the hyper-reactive protein targets and thereby increase the probability of rendering PPTs with false-negative results, while low concentrations of probe and high concentrations of incubated proteome more than likely will capture the PPTs. Based on this enlightening observation, we developed a proof-of-concept approach to identify the PPTs of iodoacetamide, a thiol-reactive MBM. This study will deepen our understanding of the enrichment process and improve the accuracy of pull-down-guided target identification

Concentration-Dependent Enrichment Identifies Primary Protein Targets of Multitarget Bioactive Molecules

Multitarget bioactive molecules (MBMs) are of increasing importance in drug discovery as they could produce high efficacy and a low chance of resistance. Several advanced approaches of quantitative proteomics were developed to accurately identify the protein targets of MBMs, but little study has been carried out in a sequential manner to identify primary protein targets (PPTs) of MBMs. This set of proteins will first interact with MBMs in the temporal order and play an important role in the mode of action of MBMs, especially when MBMs are at low concentrations. Herein, we describe a valuable observation that the result of the enrichment process is highly dependent on concentrations of the probe and the proteome. Interestingly, high concentrations of probe and low concentrations of incubated proteome will readily miss the hyper-reactive protein targets and thereby increase the probability of rendering PPTs with false-negative results, while low concentrations of probe and high concentrations of incubated proteome more than likely will capture the PPTs. Based on this enlightening observation, we developed a proof-of-concept approach to identify the PPTs of iodoacetamide, a thiol-reactive MBM. This study will deepen our understanding of the enrichment process and improve the accuracy of pull-down-guided target identification

Specific Light-Up Probe with Aggregation-Induced Emission for Facile Detection of Chymase

Human chymases are important proteases abundant in mast cell granules. The elevated level of chymases and other serine proteases is closely related to inflammatory and immunoregulatory functions. Monitoring of the chymase level is very important, however, the existing methods remain limited and insufficient. In this work, a light-up probe of TPETH-2­(CFTERD₃) (where CFTERD is Cys-Phe-Thr-Glu-Arg-Asp) was developed for chymase detection. The probe has low fluorescent signal in aqueous media, but its solubility can be changed after hydrolysis by chymase, giving significant fluorescence turn-on with a high signal-to-noise (S/N) ratio. The probe has excellent selectivity to chymase compared to other proteins and can effectively differentiate chymase from other enzymes (e.g., chymotrypsin and trypsin) in the same family (E.C. 3.4.21). The detection limit is calculated to be 0.1 ng/mL in PBS buffer with a linear range of 0–9.0 ng/mL. A comparison study using TPETH-2­(CFTERD₂) as the probe reveals the importance of molecular design in realizing the high S/N ratio. TPETH-2­(CFTERD₃) thus represents a simple turn-on probe for chymase detection, with real-time and direct readout and also excellent sensitivity and selectivity

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

Aggregation-Induced Emission Probe for Specific Turn-On Quantification of Soluble Transferrin Receptor: An Important Disease Marker for Iron Deficiency Anemia and Kidney Diseases

Transferrin receptor (TfR) is overexpressed on the surface of many cancer cells due to its vital roles in iron circulation and cellular respiration. Soluble transferrin receptor (sTfR), a truncated extracellular form of TfR in serum, is an important marker of iron deficiency anemia (IDA) and bone marrow failure in cancer patients. More recently, sTfR level in urine has been related to a specific kidney disease of Henoch–Schönlein purpura nephritis (HSPN). Despite the universal significance of sTfR, there is still a lack of a simple and sensitive method for the quantification of sTfR. Furthermore, it is desirable to have a probe that can detect both TfR and sTfR for further comparison study. In this work, we developed a water-soluble AIE–peptide conjugate with aggregation-induced emission (AIE) characteristics. Taking advantage of the negligible emission from molecularly dissolved tetraphenylethene (TPE), probe TPE-2T7 was used for the light-up detection of sTfR. The probe itself is nonemissive in aqueous solution, but it turns on its fluorescence upon interaction with sTfR to yield a detection limit of 0.27 μg/mL, which is much lower than the sTfR level in IDA patients. Furthermore, a proof-of-concept experiment validates the potential of the probe for diagnosis of HSPN by urine test

Small Molecule Probe Suitable for <i>In Situ</i> Profiling and Inhibition of Protein Disulfide Isomerase

Proper folding of cellular proteins is assisted by protein disulfide isomerases (PDIs) in the endoplasmic reticulum of mammalian cells. Of the at least 21 PDI family members known in humans, the 57-kDa PDI has been found to be a potential therapeutic target for a variety of human diseases including cancer and neurodegenerative diseases. Consequently, small molecule PDI-targeting inhibitors have been actively pursued in recent years, and thus far, compounds possessing moderate inhibitory activities (IC₅₀ between 0.1 and 100 μM against recombinant PDI) have been discovered. In this article, by using <i>in situ</i> proteome profiling experiments in combination with <i>in vitro</i> PDI enzymatic inhibition assays, we have discovered a phenyl vinyl sulfonate-containing small molecule (<b>P1</b>; shown) as a relatively potent and specific inhibitor of endogenous human PDI in several mammalian cancer cells (e.g., GI₅₀ ∼ 4 μM). It also possesses an IC₅₀ value of 1.7 ± 0.4 μM in an <i>in vitro</i> insulin aggregation assay. Our results indicate <b>P1</b> is indeed a novel, cell-permeable small molecule PDI inhibitor, and the electrophilic vinyl sulfonate scaffold might serve as a starting point for future development of next-generation PDI inhibitors and probes