Hydrolysis of 5-methylfuran-2-yl to 2,5-dioxopentanyl allows for stable bio-orthogonal proximity-induced ligation

Ligation methodologies featuring bio-orthogonal units and leading to the formation of a stable adduct are the ideal candidates for being applied in a biological context. However, most of the available strategies rely on highly reactive species that require careful handling, or on the activation of pro-reactive functional groups. We here report on a proximity-induced ligation reaction that relies on a stable 2,5-dione, that can be conveniently generated under acidic conditions from a 2,5-dialkylfuran building block, and hydrazine nucleophiles. This bio-orthogonal ligation, which proceeds under physiological conditions, does not require any stimulus or trigger and leads to the formation of a pyridazinium adduct that demonstrates excellent stability under harsh conditions (24 h at 90 °C). The reaction was applied to the formation of PNA-PNA adducts, DNA- and RNA-templated ligations, and for the formation of peptide-peptide adducts in solution. This convenient methodology was further implemented on plastic and glass surfaces to realize self-addressable covalent constructs.

Computational prediction of photosensitizers’ toxicity

The percentage of failures in late pharmaceutical development due to toxicity has increased dramatically over the last decade or so, resulting in increased demand for new methods to rapidly and reliably predict the toxicity of compounds. Today, computational toxicology can be used in every phase of drug discovery and development, from profiling large libraries early on, to predicting off-target effects in the mid-discovery phase, and to assess potential mutagenic impurities in development and degradants as part of life-cycle management. In this study, for the first time, in silico approaches were used to analyze the possible dark toxicity of photosensitive systems based on chlorin e6 and assessed possible toxicity of these compositions. By applying quantitative structure-activity relationship models (QSARs) and modeling adverse outcome pathways (AOPs), a potential toxic effect of water-soluble (chlorin e6 and chlorin e6 aminoamid) and hydrophobic (tetraphenylporphyrin) photosensitizers (PS) was predicted. Particularly, PSs’ protein binding ability, reactivity to form peptide adducts, glutathione conjugation, activity in dendritic cells, and gene expression activity in keratinocytes were explored. Using a metabolism simulator, possible PS metabolites were predicted and their potential toxicity was assessed as well. It was shown that all tested porphyrin PS and their predicted metabolites possess low activity in the mentioned processes and therefore are unable to cause significant adverse toxic effects under dark conditions.

Isotypes of autoantibodies against novel differential 4-hydroxy-2-nonenal-modified peptide adducts in serum is associated with rheumatoid arthritis in Taiwanese women

Background Rheumatoid arthritis (RA) is an autoimmune disorder with systemic inflammation and may be induced by oxidative stress that affects an inflamed joint. Our objectives were to examine isotypes of autoantibodies against 4-hydroxy-2-nonenal (HNE) modifications in RA and associate them with increased levels of autoantibodies in RA patients. Methods Serum samples from 155 female patients [60 with RA, 35 with osteoarthritis (OA), and 60 healthy controls (HCs)] were obtained. Four novel differential HNE-modified peptide adducts, complement factor H (CFAH)1211–1230, haptoglobin (HPT)78–108, immunoglobulin (Ig) kappa chain C region (IGKC)2–19, and prothrombin (THRB)328–345, were re-analyzed using tandem mass spectrometric (MS/MS) spectra (ProteomeXchange: PXD004546) from RA patients vs. HCs. Further, we determined serum protein levels of CFAH, HPT, IGKC and THRB, HNE-protein adducts, and autoantibodies against unmodified and HNE-modified peptides. Significant correlations and odds ratios (ORs) were calculated. Results Levels of HPT in RA patients were greatly higher than the levels in HCs. Levels of HNE-protein adducts and autoantibodies in RA patients were significantly greater than those of HCs. IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgM anti-IGKC2−19 HNE may be considered as diagnostic biomarkers for RA. Importantly, elevated levels of IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgG anti-THRB328−345 were positively correlated with the disease activity score in 28 joints for C-reactive protein (DAS28-CRP). Further, the ORs of RA development through IgM anti-HPT78−108 HNE (OR 5.235, p < 0.001), IgM anti-IGKC2−19 (OR 12.655, p < 0.001), and IgG anti-THRB328−345 (OR 5.761, p < 0.001) showed an increased risk. Lastly, we incorporated three machine learning models to differentiate RA from HC and OA, and performed feature selection to determine discriminative features. Experimental results showed that our proposed method achieved an area under the receiver operating characteristic curve of 0.92, which demonstrated that our selected autoantibodies combined with machine learning can efficiently detect RA. Conclusions This study discovered that some IgG- and IgM-NAAs and anti-HNE M-NAAs may be correlated with inflammation and disease activity in RA. Moreover, our findings suggested that IgM anti-HPT78−108 HNE, IgM anti-IGKC2−19, and IgG anti-THRB328−345 may play heavy roles in RA development.

Mass Spectrometry-Based Solid Phase Peptide Reaction Assay for Detecting Allergenicity Using an Immobilized Peptide-Conjugating Photo-Cleavable Linker

The biological process of skin sensitization depends on the ability of a sensitizer to modify endogenous proteins. A direct peptide reactivity assay (DPRA), based on the biological process of skin sensitization, was developed as an alternative to controversial animal experiments. Although DPRA has been endorsed by industries and is internationally accepted as promising, it has several drawbacks, such as incompatibility with hydrophobic chemicals, inability to perform detailed reaction analysis, and ability to evaluate only single components. Here, we demonstrated that sensitizers and peptide adducts can be easily identified using a mass spectrometry-based solid-phase peptide reaction assay (M-SPRA). We synthesized peptides with a photo-cleavable linker immobilized on resins. We showed the potential of M-SPRA in predicting skin sensitization by measuring the peptide adducts that were selectively eluted from the resin after cleaving the linker post-reaction. M-SPRA provides more detailed information regarding chemical reactivity and accurate assessment of test samples, including mixtures. M-SPRA may be helpful for understanding the binding mechanism of sensitizers (toxicology), which may assist in further refining reactivity assays and aiding in the interpretation of reactivity data.