5-N-Carboxyimino-6-aminopyrimidine-2,4(3H)-dione, a novel indicator for hypochlorite formation.

Uric acid is an adequate and endogenous probe for identifying reactive oxygen or nitrogen species generated in vivo because its oxidation products are specific to reacted reactive oxygen or nitrogen species. Recently, we identified 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione as a hypochlorite-specific oxidation product. 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione was anticipated to be a biomarker for hypochlorite production in vivo. However, while it was stable in aqueous solution at weak acidic and alkaline pH (6.0-8.0), it was unstable in human plasma. In this study, we found that 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione rapidly reacted with thiol compounds such as cysteine and glutathione to yield 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione, which was stable in human plasma unlike 5-N-carboxyimino-6-N-chloroaminopyrimidine-2,4(3H)-dione. 5-N-carboxyimino-6-aminopyrimidine-2,4(3H)-dione was produced upon uric acid degradation during myeloperoxidase-induced uric acid oxidation and lipopolysaccharide-induced pseudo-inflammation in collected 2,4(3H)-dione has potential as a marker for hypochlorite production in vivo.

Comprehensive engineering of a therapeutic neutralizing antibody targeting SARS-CoV-2 spike protein to neutralize escape variants.

The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

4-Cl-edaravone and (E)-2-chloro-3-[(E)-phenyldiazenyl]-2-butenoic acid are the specific reaction products of edaravone with hypochlorite.

3-Methyl-1-phenyl-2-pyrazolin-5-one (edaravone) is a synthetic one-electron antioxidant used as a drug for treatment against acute phase cerebral infarction in Japan. This drug also reacts with two-electron oxidants like peroxynitrite to give predominantly 4-nitrosoedaravone but no one-electron oxidation products. It is believed that this plays a significant role in amelioration of amyotrophic lateral sclerosis. The drug was approved for treatment of amyotrophic lateral sclerosis in Japan and USA in 2015 and 2017, respectively. In this study, we examined the reaction of edaravone with another two-electron oxidant, hypochlorite anion (ClO-). Edaravone reacted with ClO- in 50% methanolic phosphate buffer (pH 7.4) solution containing typical two-electron reductants, such as glutathione, cysteine, methionine, and uric acid, as internal references. The concentration of edaravone decreased at a similar rate as each co-existing reference, indicating that it showed comparable reactivity toward ClO- as those references. Furthermore, 4-Cl-edaravone and (E)-2-chloro-3-[(E)-phenyldiazenyl]-2-butenoic acid (CPB) were identified as primary and end products, respectively, and no one-electron oxidation products were detected. These results suggest that edaravone treatment can bring greater benefit against ClO--related injury such as inflammation, and 4-Cl-edaravone and CPB can be good biomarkers for ClO--induced oxidative stress.

Defining the antibody cross-reactome directed against the influenza virus surface glycoproteins.

Infection with influenza virus induces antibodies to the viral surface glycoproteins hemagglutinin and neuraminidase, and these responses can be broadly protective. To assess the breadth and magnitude of antibody responses, we sequentially infected mice, guinea pigs and ferrets with divergent H1N1 or H3N2 subtypes of influenza virus. We measured antibody responses by ELISA of an extensive panel of recombinant glycoproteins representing the viral diversity in nature. Guinea pigs developed high titers of broadly cross-reactive antibodies; mice and ferrets exhibited narrower humoral responses. Then, we compared antibody responses after infection of humans with influenza virus H1N1 or H3N2 and found markedly broad responses and cogent evidence for 'original antigenic sin'. This work will inform the design of universal vaccines against influenza virus and can guide pandemic-preparedness efforts directed against emerging influenza viruses.