Switching of Photocatalytic Tyrosine/Histidine Labeling and Application to Photocatalytic Proximity Labeling.

Weak and transient protein interactions are involved in dynamic biological responses and are an important research subject; however, methods to elucidate such interactions are lacking. Proximity labeling is a promising technique for labeling transient ligand-binding proteins and protein-protein interaction partners of analytes via an irreversible covalent bond. Expanding chemical tools for proximity labeling is required to analyze the interactome. We developed several photocatalytic proximity-labeling reactions mediated by two different mechanisms. We found that numerous dye molecules can function as catalysts for protein labeling. We also identified catalysts that selectively modify tyrosine and histidine residues and evaluated their mechanisms. Model experiments using HaloTag were performed to demonstrate photocatalytic proximity labeling. We found that both ATTO465, which catalyzes labeling by a single electron transfer, and BODIPY, which catalyzes labeling by singlet oxygen, catalyze proximity labeling in cells.

The modified IL-8 Luc assay, an in vitro skin sensitisation test, can significantly improve the false-negative judgment of lipophilic sensitizers with logKow values > 3.5.

False-negative judgment due to poor chemical solubility is a problem with in vitro skin sensitisation tests. Water-insoluble chemicals are typically dissolved in DMSO in most sensitisation tests but precipitate when diluted with medium beyond their solubility in water. Such tests lack procedures to rule out false-negative judgments due to poor solubility. The IL-8 Luc assay (OECD442E) is unique in that if chemicals do not dissolve at 20 mg/mL in medium and have no effect on IL-8 luciferase activity (IL8LA), they are classified as indeterminate. The purpose of the present study was to reduce the number of indeterminate chemicals and improve assay performance. The IL-8 Luc assay can simultaneously examine glyceraldehyde 3-phosphate dehydrogenase luciferase activity (GAPLA) and IL8LA, and thus we examined the correlation between the reduction of GAPLA (defined as Inh-GAPLA) and the reduction of propidium iodide (PI)-excluding cells for three sensitizers and three non-sensitizers. We observed a significant correlation between luciferase activity driven by the GAPDH promoter of THP-G8 cells and the number of viable cells. Furthermore, chemicals providing an Inh-GAPLA value below 0.8 always reduced the ratio of PI-excluding cells to less than 0.6. Using the modified criteria, indeterminate chemicals are judged as negative if they provide Inh-GAPLA values below 0.8. This modification reduced the number of indeterminate chemicals and increased specificity, highlighting the unique advantage of the IL-8 Luc assay.

The IL-1 promoter-driven luciferase reporter cell line THP-G1b can efficiently predict skin-sensitising chemicals.

IL-1 functions as an essential pro-inflammatory mediator for the sensitisation of allergic contact dermatitis (ACD). However, studies conducted to date have typically used a limited number of haptens and examined their effects only on murine ACD or murine dendritic cells (DCs). It therefore remains unclear whether IL-1α and/or IL-1ß is produced in ACD induced by haptens other than those commonly used in mouse ACD models, and whether they are essential for sensitisation leading to ACD in humans. In addition, it is unclear whether human DCs also produce IL-1α or IL-1ß after stimulation by haptens in general. Here, we first demonstrated that 10 haptens (3 extreme, 1 strong, 3 moderate and 3 weak) increased both IL-1α mRNA and IL-1ß mRNA expression by the human monocyte cell line THP-1, a commonly used surrogate of DCs in in vitro skin sensitisation tests. Next, we constructed an in vitro skin sensitisation test using a stable IL-1ß reporter cell line, THP-G1b, and evaluated whether 88 haptens and 34 non-haptens increase IL-1ß reporter activity. We found that 94% of 77 haptens evaluated after considering their applicability domain and solubility in the chosen media stimulated reporter activity. These studies demonstrated that most haptens, irrespective of their potency, increased IL-1ß mRNA expression by THP-1 cells, confirming that human DCs also produce IL-1ß after stimulation by most haptens. The luciferase assay using THP-G1b cells is thus another skin sensitisation test based on the adverse outcome pathway with reasonable performance.

Optimization of the IL-2 Luc assay for immunosuppressive drugs: a novel in vitro immunotoxicity test with high sensitivity and predictivity.

We have reported that the IL-2 Luc assay can detect the effects of chemicals on IL-2 promoter activity by using a dual reporter cell line, 2H4 cells that measure IL-2 promoter-driven luciferase activity (IL2LA) and GAPDH promoter-driven luciferase activity (GAPLA). Since the IL-2 Luc assay cannot detect immunosuppressive drugs that are antimitotic towards rapidly proliferating cells, we attempted to establish a new assay to detect these chemicals by taking advantage of the dual reporter cell properties of 2H4 cells. We first determined the optimal incubation time with drugs and the seeding cell density, and confirmed that the change in GAPLA and IL2LA levels reflects the change in cell count and IL-2 production of 2H4 cells after drug treatment. We designed the IL-2 luciferase lymphotoxicity test (IL-2 Luc LTT) to detect the antimitotic effects of chemicals by modifying the protocol and criteria of the IL-2 Luc assay. To determine the performance of the IL-2 Luc LTT and that of the combination of the IL-2 Luc LTT and the IL-2 Luc assay, we examined 46 drugs: 19 immunosuppressive drugs with different mechanisms of action, 12 anti-cancer drugs, and 15 non-immunosuppressive drugs. The performances of the IL-2 Luc LTT, the IL-2 Luc assay and their combination were 43.3%, 61.3%, and 93.3%, respectively, for sensitivity, 84.6%, 53.3%, and 50.0%, respectively, for specificity, and 55.8%, 58.7%, and 79.5%, respectively, for accuracy. These results demonstrated that the combination of these two assays is promising for the detection of immunosuppressive drugs with different mechanisms of action.

Functionalization of Human Serum Albumin by Tyrosine Click.

Human serum albumin (HSA) is a promising drug delivery carrier. Although covalent modification of Cys34 is a well-established method, it is desirable to develop a novel covalent modification method that targets residues other than cysteine to introduce multiple functions into a single HSA molecule. We developed a tyrosine-selective modification of HSA. Three tyrosine selective modification methods, hemin-catalyzed, horseradish peroxidase (HRP)-catalyzed, and laccase-catalyzed reactions were performed, and the modification efficiencies and modification sites of the modified HSAs obtained by these methods were evaluated and compared. We found that the laccase-catalyzed method could efficiently modify the tyrosine residue of HSA under mild reaction conditions without inducing oxidative side reactions. An average of 2.2 molecules of functional groups could be introduced to a single molecule of HSA by the laccase method. Binding site analysis using mass spectrometry suggested Y84, Y138, and Y401 as the main modification sites. Furthermore, we evaluated binding to ibuprofen and found that, unlike the conventional lysine residue modification, the inhibition of drug binding was minimal. These results suggest that tyrosine-residue selective chemical modification is a promising method for covalent drug attachment to HSA.

Profiling the immunotoxicity of chemicals based on in vitro evaluation by a combination of the Multi-ImmunoTox assay and the IL-8 Luc assay.

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), which can evaluate the effects of chemicals on the promoter activities of four cytokines: IL-2, IFN-γ, IL-1ß, and IL-8. We previously reported that MITA correctly reflected the change in mRNA of human whole-blood cells treated with dexamethasone, cyclosporine, FK506, or several other immunosuppressive drugs. In this study, we combined MITA with the IL-8 Luc assay to detect skin sensitization chemicals (OECD 442E) (modified MITA: mMITA) and established a data set of 60 chemicals examined by mMITA. Using the mMITA results, chemicals can be classified based on the lowest observed effect level (LOEL) of chemicals in suppressing or augmenting the promoter activities of the four cytokines. Moreover, we demonstrated that K-means clustering and hierarchical clustering of the 60 chemicals based on the LOEL for their effects on IL-2 and IL-8 promoter activities and the judgment by the IL-8 Luc assay resulted in the same 6-cluster solution: cluster 1 with preferential suppression of IL-8, cluster 2 with suppression of IL-2 and a positive IL-8 Luc assay result, cluster 3 with suppression of both IL-2 and IL-8, cluster 4 with no effects on IL-2 or IL-8 and a negative IL-8 Luc assay result, cluster 5 with suppression of both IL-2 and IL-8 and a negative IL-8 Luc assay result, and cluster 6 with preferential suppression of IL-8. These data suggest that mMITA is a promising novel high-throughput approach for detecting unrecognized immunological effects of chemicals and for profiling their immunotoxic effects.

An international validation study of the interleukin-2 luciferase leukocyte toxicity test (IL-2 Luc LTT) to evaluate potential immunosuppressive chemicals and its performance after use with the interleukin-2 luciferase assay (IL-2 Luc assay).

We previously reported that the IL-2 Luc LTT can detect immunosuppressive effects of drugs that are attributed to their antimitotic activity. Here, we report an official validation study of the IL-2 Luc LTT. In the Phase I study that evaluated five coded chemicals, the within-laboratory reproducibility of three independent laboratories was 100.0%. In the combined results of the Phase I and II studies that evaluated 20 coded chemicals, the between-laboratory reproducibility was 92.0%. When compared with the reference data based on the previously-reported immunotoxicological information, the predictivity of the combined Phase I and II studies was 76.0% for Lab A and 72.0% for Labs B and C. In contrast, in the study in which the lead laboratory examined 37 non-pharmaceutical chemicals, the predictivity of the IL-2 Luc LTT and the IL-2 Luc assay was 48.6% and 64.9%, respectively, whereas that of the combined assays was 74.3%. It is clear that an integrated approach combining multiple assays is necessary for the development of in vitro immunosuppression testing. These data suggest that the IL-2 Luc LTT alone is not sufficient as a component of the integrated approach, but the combination of the IL-2 Luc assay and IL-2 Luc LTT is promising.

Development of a novel in vitro assay to evaluate environmental water using an IL-8 reporter cell line.

The IL-8 luciferase reporter cell line, THP-G8 cells, used in the in vitro sensitization test, OECD442E, can respond to a variety of stimuli other than haptens, such as lipopolysaccharide (LPS), other bacterial toxins, and detergents. Considering these characteristics, we examined the ability of the IL-8 luciferase assay using THP-G8 cells to evaluate water pollution. We first stimulated THP-G8 cell with various Toll-like receptor (TLR) agonists and nucleotide-binding oligomerization domain-like receptor (NLR) agonists, and found that TLR1, 2, 4, 5, 6 agonists and NOD 1, 2 agonists significantly augmented IL-8 luciferase activity (IL8LA). Then, we examined the detection threshold of LPS by THP-G8 cells, and found it 0.4 EU/ml. Next, we examined whether THP-G8 cells can differently respond to a variety of sources of environmental water around Sendai, Japan and Manila, Philippine and whether there is a correlation between the IL8LA of different sources of water and their level of endotoxin assessed by the LAL assay. There was a clear trend that the IL8LA was lower in the upper stream and higher in the downstream in both Japan and Philippine. Moreover, there was a strong correlation between the IL8LA of the environmental water and its endotoxin level. Finally, using N-acetyl-L-cysteine, an antioxidant/radical scavenger, and polymyxin B that neutralizes endotoxin, we demonstrated that there was a difference in the suppressive effects by them between the water from Japan and that from Philippine. These data suggest the potential of the IL-8 luciferase assay for evaluating environmental water pollution both quantitatively and qualitatively.

An international validation study of the IL-2 Luc assay for evaluating the potential immunotoxic effects of chemicals on T cells and a proposal for reference data for immunotoxic chemicals.

To evaluate the immunotoxic effects of xenobiotics, we have established the Multi-ImmunoTox assay, in which three stable reporter cell lines are used to evaluate the effects of chemicals on the IL-2, IFN-γ, IL-1ß and IL-8 promoters. Here, we report the official validation study of the IL-2 luciferase assay (IL-2 Luc assay). In the Phase I study that evaluated five coded chemicals in three sets of experiments, the average within-laboratory reproducibility was 86.7%. In the Phase II study, 20 coded chemicals were evaluated at multiple laboratories. In the combined results of the Phase I and II studies, the between-laboratory reproducibility was 80.0%. These results suggested that the IL-2 Luc assay was reproducible both between and within laboratories. To determine the predictivity, we collected immunotoxicological information and constructed the reference data by classifying the chemical into immunotoxic compounds targeting T cells or others according to previously reported criteria. When compared with the reference data, the average predictivity of the Phase I and II studies was 75.0%, while that of additional 60 chemicals examined by the lead laboratory was 82.5%. Although the IL-2 Luc assay alone is not sufficient to predict immunotoxicity, it will be a useful tool when combined with other immune tests.

The performance of an in vitro skin sensitisation test, IL-8 Luc assay (OECD442E), and the integrated approach with direct peptide reactive assay (DPRA).

In all current in vitro skin sensitisation assays, DMSO is used to dissolve water-insoluble chemicals. However, our previous study suggested the superiority of the modified IL-8 Luc assay (mIL-8 Luc), in which X-VIVOTM 15 is used to dissolve chemicals, over the original assay using DMSO (oIL-8 Luc). In this study, to confirm the superiority of the mIL-8 Luc, we first increased the number of chemicals examined and demonstrated the superiority of the mIL-8 Luc, in which the mIL-8 Luc provided 87.6% of sensitivity, 74.2% of specificity, and 84.6% of accuracy. Next, to clarify the cause of false negative judgment by the mIL-8 Luc, we examined the effects of physical properties of chemicals on judgment. The results demonstrated that high molecular weight, high LogKo/w, or poor water solubility, did not cause false negative judgment. When it was accepted as an OECD test guideline, the criteria of the mIL-8 Luc to determine sensitisers were modified to further decrease false negative judgment by poor solubility. By applying the new criteria, the test guideline IL-8 Luc assay (tgIL-8 Luc) improved sensitivity but decreased specificity and increased the number of chemicals that cannot be judged. To overcome this problem, we examined a simple combination of the tgIL-8 Luc with direct peptide reactive assay (DPRA), which could improve specificity and decrease the number of the chemicals that cannot be judged. These data suggest that the tgIL-8 Luc is a promising in vitro skin sensitisation assay in combination with other in vitro or in chemico methods.

Optimization of the IL-8 Luc assay as an in vitro test for skin sensitization.

We previously reported a dataset of the IL-8 Luc assay covering reference chemicals published by ECVAM, in which the effects of chemicals on IL-8 promoter activity were evaluated by an IL-8 reporter cell line, THP-G8 cells. To clarify its performance, we created another dataset of 88 sensitizers and 34 non-sensitizers. Simultaneously, to improve its performance, we changed the incubation time from 5 h to 16 h, deleted the criterion regarding the effects of N-acetylcysteine, and set an exclusion criterion for detergents. These modifications significantly improved its performance. In addition, we examined the following three criteria to judge chemicals as sensitizers: Criterion 1: Fold induction of SLO luciferase activity (FlnSLO-LA)⩾1.4, Criterion 2: the lower limit of the 95% confidence interval of FInSLO-LA⩾1.0, Criterion 3: the intersection of criteria 1 and 2. Among them, Criterion 1 produced the best performance, demonstrating that the accuracy, sensitivity and specificity were 81%, 79%, and 90%, respectively. In addition, we found that the IL-8 Luc assay solubilizing chemicals with X-VIVO substantially improved its performance. Finally, the IL-8 Luc assay combined with DPRA and DEREK could improve substantially its performance. These data suggest that the IL-8 Luc assay is a promising test method to screen skin sensitizers.

Evaluation of the Multi-ImmunoTox Assay composed of 3 human cytokine reporter cells by examining immunological effects of drugs.

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), to evaluate the effects on key predictivein vitro components of the human immune system. The system is composed of 3 stable reporter cell lines transfected with 3 luciferase genes, SLG, SLO, and SLR, under the control of 4 cytokine promoters, IL-2, IFN-γ, IL-1ß, and IL-8, and the G3PDH promoter. We first compared the effects of dexamethasone, cyclosporine, and tacrolimus on these cell lines stimulated with phorbol 12-myristate 13-acetate and ionomycin, or lipopolysaccharides, with those on mRNA expression by the mother cell lines and human whole blood cells after stimulation. The results demonstrated that MITA correctly reflected the change of mRNA of the mother cell lines and whole blood cells. Next, we evaluated other immunosuppressive drugs, off-label immunosuppressive drugs, and non-immunomodulatory drugs. Although MITA did not detect immunosuppressive effects of either alkylating agents or antimetabolites, it could demonstrate those of the off-label immunosuppressive drugs, sulfasalazine, chloroquine, minocycline, and nicotinamide. Compared with the published immunological effects of the drugs, these data suggest that MITA can present a novel high-throughput approach to detect immunological effects of chemicals other than those that induce immunosuppressive effects through their inhibitory action on cell division.