Photodynamic Therapy by Glucose Transporter 1-Selective Light Inactivation.

Chromophore-assisted light inactivation (CALI) was applied to molecule-targeted photodynamic therapy (PDT). In order to identify organic photosensitizers suitable for CALI, the carbonic anhydrase II (CAII) ligand, 4-sulfamoylbenzoic acid 1, was conjugated with several photosensitizers to produce compounds 2-7, whose CALI ability was evaluated by measuring their effect on CAII enzymatic activity. Di-iodinated BODIPY (I2BODIPY) exhibited excellent CAII inactivation ability, similar to that of Ru(bpy)3. The glucose-I2BODIPY conjugate (8) was synthesized as an inactivation of glucose transporter 1 (GLUT1), a protein overexpressed in many cancer cells. Under light irradiation, 8 exhibited concentration-dependent cytotoxicity with half maximal inhibitory concentration (IC50) values of 5.49, 11.14, and 8.73 µM, against human cervical carcinoma (HeLa), human lung carcinoma (A549), and human hepatocellular carcinoma (HepG2) cell lines, respectively. The GLUT1 inhibitor phloretin suppressed the cytotoxicity induced by 8 under light irradiation in a concentration-dependent manner. Western blot analysis indicated that GLUT1 was not detected in cell lines treated with 10 µM 8 under light irradiation. Furthermore, 8 reduced the levels of epidermal growth factor receptor tyrosine kinase (EGFR), phospho-ERK (Y204), and GLUT1 without affecting ERK, α-tubulin, and PCNA protein levels, whereas talaporfin sodium, a clinically approved photosensitizer for PDT, nonspecifically reduced intracellular protein levels in HeLa cells, indicating that 8 has a GLUT1-specific inactivation ability and causes light-induced cytotoxicity by modulating the EGFR/MAPK signaling pathway.

Intracellular photocatalytic-proximity labeling for profiling protein-protein interactions in microenvironments.

Intracellular photocatalytic-proximity labeling (iPPL) was developed to profile protein-protein interactions in the microenvironment of living cells. Acriflavine was found to be an efficient cell-membrane-permeable photocatalyst for introduction into the genetically HaloTag-fused protein of interest for iPPL with a radical labeling reagent, 1-methyl-4-arylurazole. iPPL was applied to the histone-associated protein H2B in HaloTag-H2B expressing HEK293FT cells. The proteins directly interacting with histones and RNA-binding proteins were selectively labeled in the intracellular environment, suggesting that the iPPL method has a smaller labeling radius (CA. 6 nm) than the BioID and APEX methods.

Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen.

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography-mass spectrometry analysis. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.

Target Protein Identification on Photocatalyst-Functionalized Magnetic Affinity Beads.

Although various affinity chromatography and photoaffinity labeling methods have been developed for target protein identification of bioactive molecules, it is often difficult to detect proteins that bind the ligand with weak transient affinity using these techniques. We have developed single electron transfer-mediated tyrosine labeling using ruthenium photocatalysts. Proximity labeling using 1-methyl-4-aryl-urazole (MAUra) labels proteins in close proximity to the photocatalyst with high efficiency and selectivity. Performing this labeling reaction on affinity beads makes it possible to label proteins that bind the ligand with weak transient affinity. In this article, novel protocols are described for target protein identification using photocatalyst proximity labeling on ruthenium photocatalyst-functionalized magnetic affinity beads. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of ruthenium photocatalyst Basic Protocol 2: Synthesis of azide- or desthiobiotin-conjugated labeling reagents Basic Protocol 3: Preparation of photocatalyst and ligand-functionalized affinity beads Basic Protocol 4: Target protein labeling in cell lysate Basic Protocol 5: Enrichment of labeled proteins with MAUra-DTB for LC-MS/MS analysis Basic Protocol 6: 2D-DIGE analysis of fluorescence-labeled proteins.

Catalyst-proximity protein chemical labelling on affinity beads targeting endogenous lectins.

Magnetic affinity beads functionalized with lactose and ruthenium/dcbpy complexes were developed. Using MAUra, a catalyst-proximity labelling reagent, the catalytic labeling of lactose-binding proteins was achieved with high selectivity on the beads. The first unbiased identification of cellular endogenous lectins bound to lactose (galectin-1 and galectin-3) was achieved with chemical labelling on the affinity beads.

Target-protein-selective inactivation and labelling using an oxidative catalyst.

Reactive oxygen species (ROS) and radical species generated using oxidative single-electron transfer (SET) catalysts are highly reactive, inducing local environmental oxidative reactions, resulting in protein inactivation and labelling in proximity to the catalysts. Oxidative catalysts bound to the target protein generate ROS which induce oxidation only within a limited radius (∼30 nm), resulting in target-protein-selective inactivation. On the other hand, protein chemical labelling reactions via ROS or SET induced by the catalysts are completed in proximity to the catalyst. These proximity labelling techniques have recently attracted considerable attention as innovative tools to elucidate protein interaction mapping and unknown protein-protein interaction (PPI) partners. Not only can peroxidases be genetically introduced into the protein of interest but also ligand-conjugated catalysts can catalyze oxidative SET reactions in a protein mixture under intracellular conditions. In this review, we focus on two approaches of selective inactivation of protein functions and selective protein labelling using oxidative SET catalysts.

1-Methyl-4-aryl-urazole (MAUra) labels tyrosine in proximity to ruthenium photocatalysts.

We designed and synthesised peptides conjugated with proline linkers and ruthenium photocatalysts. These peptides were used as substrates to evaluate the photocatalyst-proximity dependences of candidates for tyrosine labelling reagents. The 1-methyl-4-aryl-urazole (MAUra) structure was found to be a novel tyrosyl radical trapping agent to label tyrosine residues effectively under the conditions where the ruthenium photocatalyst and tyrosine were in close proximity. Using a ruthenium photocatalyst conjugated to a carbonic anhydrase ligand, the target protein in a complex protein mixture was labelled with remarkable target selectivity by azide- or desthiobiotin-conjugated MAUra derivatives.

[Development and Application of Catalytic Tyrosine Modification].

The chemical labeling of proteins with synthetic probes is a key technique used in chemical biology, protein-based therapy, and material science. Much of the chemical labeling of native proteins, however, depends on the labeling of lysine and cysteine residues. While those methods have significantly contributed to native protein labeling, alternative methods that can modify different amino acid residues are still required. Herein we report the development of a novel methodology of tyrosine labeling, inspired by the luminol chemiluminescence reaction. Tyrosine residues are often exposed on a protein's surface and are thus expected to be good targets for protein functionalization. In our studies so far, we have found that 1) hemin oxidatively activates luminol derivatives as a catalyst, 2) N-methyl luminol derivative specifically forms a covalent bond with a tyrosine residue among the 20 kinds of natural amino acid residues, and 3) the efficiency of tyrosine labeling with N-methyl luminol derivative is markedly improved by using horseradish peroxidase (HRP) as a catalyst. We were able to use molecular oxygen as an oxidant under HRP/NADH conditions. By using these methods, the functionalization of purified proteins was carried out. Because N-methyl luminol derivative is an excellent protein labeling reagent that responds to the activation of peroxidase, this new method is expected to open doors to such biological applications as the signal amplification of HRP-conjugated antibodies and the detection of protein association in combination with peroxidase-tag technology.

Selective purification and chemical labeling of a target protein on ruthenium photocatalyst-functionalized affinity beads.

Selective purification and chemical labeling of a target protein in a protein mixture were simultaneously achieved on the surface of affinity beads functionalized with ligands, such as benzenesulfonamide and methotrexate (MTX), and a ruthenium complex containing 2,2'-bipyridine-4,4'-dicarboxylic acid (dcbpy). Chemical labeling of the target protein with a tyrosine radical trapper (TRT) proceeded on the surface of the beads when the target protein was in close proximity to the ruthenium photocatalyst. Both the protein purification and chemical labeling abilities of the affinity beads functionalized with ruthenium photocatalyst were not compromised after recycling several times. Dihydrofolate reductase (DHFR) endogenously expressed in HeLa cells was detected by chemical labeling with biotin-TRT on the affinity beads with high sensitivity compared to the conventional silver staining method.