Chong-Jing Zhang

Specific Light-Up Bioprobe with Aggregation-Induced Emission and Activatable Photoactivity for the Targeted and Image-Guided Photodynamic Ablation of Cancer Cells†

Activatable photosensitizers (PSs) have been widely used for the simultaneous fluorescence imaging and photodynamic ablation of cancer cells. However, the ready aggregation of traditional PSs in aqueous media can lead to fluorescence quenching as well as reduced phototoxicity even in the activated form. We have developed a series of PSs that show aggregation-enhanced emission and phototoxicity and thus the exact opposite behavior to that of previously reported PSs. We further developed a dual-targeted enzyme-activatable bioprobe based on the optimized photosensitizer and describe simultaneous light-up fluorescence imaging and activated photodynamic therapy for specific cancer cells. The design of smart probes should thus open new opportunities for targeted and image-guided photodynamic therapy.

Haem-activated promiscuous targeting of artemisinin in Plasmodium falciparum

The mechanism of action of artemisinin and its derivatives, the most potent of the anti-malarial drugs, is not completely understood. Here we present an unbiased chemical proteomics analysis to directly explore this mechanism in Plasmodium falciparum. We use an alkyne-tagged artemisinin analogue coupled with biotin to identify 124 artemisinin covalent binding protein targets, many of which are involved in the essential biological processes of the parasite. Such a broad targeting spectrum disrupts the biochemical landscape of the parasite and causes its death. Furthermore, using alkyne-tagged artemisinin coupled with a fluorescent dye to monitor protein binding, we show that haem, rather than free ferrous iron, is predominantly responsible for artemisinin activation. The haem derives primarily from the parasites haem biosynthesis pathway at the early ring stage and from haemoglobin digestion at the latter stages. Our results support a unifying model to explain the action and specificity of artemisinin in parasite killing.

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 (DA-2) 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 DA-2 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.

A Photoactivatable AIE Polymer for Light-Controlled Gene Delivery: Concurrent Endo/Lysosomal Escape and DNA Unpacking†

Endo/lysosomal escape of gene vectors and the subsequent unpacking of nucleic acids in cytosol are two major challenges for efficient gene delivery. Herein, we report a polymeric gene delivery vector, which consists of a photosensitizer (PS) with aggregation-induced emission (AIE) characteristics and oligoethylenimine (OEI) conjugated via an aminoacrylate (AA) linker that can be cleaved by reactive oxygen species (ROS). In aqueous media, the polymer could self-assemble into bright red fluorescent nanoparticles (NPs), which can efficiently bind to DNA through electrostatic interaction for gene delivery. Upon visible light irradiation, the generated ROS can break the endo/lysosomal membrane and the polymer, resulting in light-controlled endo/lysosomal escape and unpacking of DNA for efficient gene delivery. The smart polymer represents the first successful gene vector to simultaneously address both challenges with a single light excitation process.

Mapping sites of aspirin-induced acetylations in live cells by quantitative acid-cleavable activity-based protein profiling (QA-ABPP)

Target-identification and understanding of mechanism-of-action (MOA) are challenging for development of small-molecule probes and their application in biology and drug discovery. For example, although aspirin has been widely used for more than 100 years, its molecular targets have not been fully characterized. To cope with this challenge, we developed a novel technique called quantitative acid-cleavable activity-based protein profiling (QA-ABPP) with combination of the following two parts: (i) activity-based protein profiling (ABPP) and iTRAQ™ quantitative proteomics for identification of target proteins and (ii) acid-cleavable linker-based ABPP for identification of peptides with specific binding sites. It is known that reaction of aspirin with its target proteins leads to acetylation. We thus applied the above technique using aspirin-based probes in human cancer HCT116 cells. We identified 1110 target proteins and 2775 peptides with exact acetylation sites. By correlating these two sets of data, 523 proteins were identified as targets of aspirin. We used various biological assays to validate the effects of aspirin on inhibition of protein synthesis and induction of autophagy which were elicited from the pathway analysis of Aspirin target profile. This technique is widely applicable for target identification in the field of drug discovery and biology, especially for the covalent drugs.

One- and Two-Photon Live Cell Imaging Using a Mutant SNAP-Tag Protein and Its FRET Substrate Pairs

A small molecule-assisted protein labeling strategy based on a mutant SNAP-Tag (mSNAP) and its FRET substrate pairs has been developed. Both one- and two-photon fluorescence microscopic experiments were successfully demonstrated in living cells.

Dual-targeted activatable photosensitizers with aggregation-induced emission (AIE) characteristics for image-guided photodynamic cancer cell ablation

The currently available photosensitizers (PSs) for photodynamic therapy (PDT) can easily lead to undesirable normal cell death due to their intrinsic photo-toxicity and lack of selectivity for cancer cells. Activatable PSs with high therapeutic efficiency towards cancer cells but minimized side effects on normal cells are thus highly desirable. In this work, we developed a probe with dual-targeted activatable PSs that can recognize and ablate cancer cells with high selectivity. The probe is composed of a fluorophore with aggregation-induced emission (AIE) characteristics which can be used as an imaging agent as well as a PS, a quencher moiety that can be cleaved upon encountering biothiols, and a cyclic arginine-glycine-aspartic acid (cRGD) tripeptide for targeting cancer cells with overexpressed αvβ3 integrin. The probe itself is non-fluorescent and its ability to generate reactive oxygen species (ROS) is prohibited. However, it could be selectively activated to offer specific fluorescence turn-on with efficient ROS generation in the aggregated state, which was used to ablate cancer cells overexpressing both αvβ3 integrin receptors and glutathione. As compared to conventional activatable PSs which show quenched fluorescence and reduced ROS generation in the aggregated state, the dual-selection process with enhanced fluorescence and efficient ROS generation of the activated AIE probe in aggregated state offers a high signal-to-background ratio for MDA-MB-231 cancer cell imaging and ablation. This strategy thus opens up new opportunities for designing activatable PSs with high selectivity and low intrinsic photo-toxicity for photodynamic cancer cell ablation.

In situ Proteomic Profiling of Curcumin Targets in HCT116 Colon Cancer Cell Line

To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQTM quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway AnalysisTM (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death.

Preparation of Small-Molecule Microarrays by trans-Cyclooctene Tetrazine Ligation and Their Application in the High-Throughput Screening of Protein–Protein Interaction Inhibitors of Bromodomains†

The e-N-acetylation of lysine residues (Kac) is one of the most common posttranslational modifications in proteins that are associated with epigenetics, and frequently occurs in large macromolecular complexes that play a role in chromatin remodelling, DNA damage, and cell-cycle control. In histones, acetylated lysines reduce electrostatic interactions with the negatively charged DNA phosphates, thus providing a more relaxed chromatin structure, which is associated with transcriptionally active genes. The cellular histone acetylation levels are strictly controlled by histone acetyltransferases (HATs; epigenetic “writers”) and histone deacetylases (HDACs; epigenetic “erasers”). 3] Furthermore, Kac affects gene transcription through interactions with bromodomain (BRD)-containing proteins (BCPs; epigenetic “readers”). HATs and HDACs have traditionally been the main research focus in medicinal chemistry and drug discovery for epigenetic diseases. As enzymes, they are considered as “druggable”. The development of protein–protein interaction (PPI) inhibitors that target the epigenetic readers, on the other hand, is much more challenging. It is generally believed that small molecules do not bind sufficiently tightly to the much larger protein–protein interface because they contain only a limited number of functional groups. Such simplistic views, however, have recently been challenged by two ground-breaking studies that showed that certain benzodiazepine-containing compounds (e.g., JQ1 and I-BET) are specific nanomolar PPI inhibitors of BET bromodomains (e.g., BRD4). This has spurred interest in compounds that might possess similar biological activities against other BRDs. However, there remains a lack of sensitive assays for rapid and high-throughput screening (HTS) of other BRD-binding compounds. Existing assays, including those based on fluorescence polarization (FP), isothermal titration calorimetry (ITC), protein stability shift, and surface plasmon resonance (SPR), are either applicable to only a few wellknown BRDs (e.g., BRD2/3/ 4), or they suffer from limited throughput because large amounts of proteins are required. Furthermore, for most of the 61 human BRDs, their cognate Kac-binding sequences are not well-understood. Recent large-scale structural studies and histone–peptide membrane arrays failed to identify any sub-micromolar-binding acetylated peptides that could be suitable for HTS assays. It is perhaps not surprising that both JQ1 and I-BET were serendipitously discovered by HTS of random in-house compound libraries using cell-based reporter assays without any prior target information. These assays, though useful in their own right, are not target-oriented, and compounds identified during these assays may not actually act on their intended BRDs. Therefore, a prerequisite for drug-development efforts that focus on the discovery of new BRD inhibitors is the development of an HTS platform that is capable of general, rapid, and systematic large-scale screening of the interactions between BRDs and small molecules. We recently developed a powerful small-molecule microarray (SMM) that is capable of sensitive, quantitative, and rapid identification of cell-permeable small-molecule PPI inhibitors. SMMs are miniaturized assemblies of small molecules that are immobilized across a planar glass slide, on which thousands of protein–ligand interactions (strong and weak) may be measured, with minimal consumption of proteins and ligands (in the nL to mL range). We reasoned that BRDs, which are protein-binding domains that normally bind to cognate acetylated peptides with moderate or weak affinities, would be well-suited for our SMM approach. Herein, in a proof-of-concept study, we have successfully constructed a SMM on which 48 different benzodiazepines that are based on the core structure of JQ 1/I-BET were immobilized, and screened it against 55 fluorescently labeled proteins, twelve of which were human BRDs (Figure 1). Specifically, we have 1) developed the first SMM that is based on bioorthogonal trans-cyclooctene (TCO) tetrazine ligation for site-specific covalent immobilization of TCO-modified benzodiazepines with unprecedented speed; 2) used this SMM for miniaturized HTS of these compounds against [*] C.-J. Zhang, C. Y. J. Tan, Dr. J. Ge, Z. Na, G. Y. J. Chen, Dr. M. Uttamchandani, Prof. Dr. S. Q. Yao Department of Chemistry, National University of Singapore 3 Science Drive 3, Singapore 117543 (Singapore) E-mail: chmyaosq@nus.edu.sg Homepage: http://staff.science.nus.edu.sg/~ syao

AIEgens for Real-time Naked-eye Sensing of Hydrazine in Solution and on a Paper Substrate: Structure-dependent Signal Output and Selectivity

Paper-based assay is a promising alternative sensing technology due to its portability, low cost and ease of operation compared to the solution sensing method. Most of current fluorophores suffer from aggregation-caused quenching, which affects their signal output in the solid state. Although fluorogens with aggregation-induced emission (AIEgens) have attracted intense research interest for solution assays, they have been rarely employed for solid phase detection due to their high emissivity in the aggregated state. In this work, three fluorogens TPE-DCV, MTPE-DCV and NTPE-DCV were designed and synthesized by the integration of intramolecular charge transfer and AIE characteristics to fine-tune their absorption and emission maxima. Among the three AIEgens, NTPE-DCV gives the best response to hydrazine, with a detection limit of 143 ppb in solution. In addition, the NTPE-DCV stained paper strip offers fluorescence turn-on from dark to yellow for 1 mM hydrazine solution or 1% hydrazine vapor for naked-eye sensing. It was also found that the fluorogen with a stronger electron donor (e.g.NTPE-DCV) showed better selectivity to hydrazine over glutathione. The practical example of hydrazine detection elucidates a general strategy for the design of AIE probes that are compatible with both solution and paper-based assays with a high sensitivity and rapid signal readout.