Infectious disease biomarkers

Abstract

To realize the usefulness of a sulfur metabolite based on active sulfur metabolomics.SOLUTION: A biomarker for the diagnosis of an infection disease is a sulfur metabolite. The sulfur metabolite may exist in expired air and the sulfur metabolite can be detected from a condensate liquid of the expired air. The biomarker of an infection disease includes: the biomarker for diagnosing bacterial and viral injections, especially, a COVID-19 infection; the biomarker for diagnosing pneumonia such as infectious pneumonia, especially, COVID-19 infectious interstitial pneumonia, and other alveolar pneumonia; and the biomarker for diagnosing an infectious exacerbation risk, especially, a risk of a shift to the exacerbation of COVID-19. There is provided a diagnostic system for detecting the infection diagnostic biomarker comprising the sulfur metabolite from the expired air, and also there is provided a system for determining an infection, especially, diagnosing the COVID-19 infection.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to biomarkers of infectious diseases and relates to sulfur metabolites. [Background technology]

[0002] The inventors have been elucidating the physiological functions and metabolic pathways of in vivo reactive sulfur species (RSS), such as cysteine hydropolysulfide (CysSSH) and glutathione polysulfide (GSSH) (Non-Patent Documents 1 and 2).

[0003] Cysteine hydropolysulfides (CysSSHs) occur abundantly in a variety of organisms, yet little is known about their biosynthesis and physiological functions. Extensive persulfide formation is evident in cysteine-containing proteins in Escherichia coli and mammalian cells, which is thought to result from post-translational processes involving chemical reactions related to sulfur metabolites in vivo.

[0004] There is efficient CysSSH synthesis from the substrate L-cysteine, a reaction catalyzed by cysteinyl-tRNA synthetase (CARS) in prokaryotes and mammals. Targeted disruption of the gene encoding mitochondrial CARS in mouse and human cells indicates that CARS plays a critical role in endogenous CysSSH production, suggesting that these enzymes function as major cysteine persulfur synthases in vivo. CARS also catalyzes cotranslational cysteine persulfation and is involved in regulating mitochondrial biogenesis and bioenergetics.

[0005] Therefore, elucidating the mechanism of CARS-dependent persulfated metabolite production may provide insight into abnormal redox signaling in physiological and pathophysiological conditions and suggest therapeutic targets based on oxidative stress and mitochondrial dysfunction.

[0006] The chemical properties of polysulfides are not fully understood and elucidated due to their reactivity or complex redox activity. However, the inventors have developed an active sulfur metabolomics analysis using RSS metabolic profiling, which is revealing the in vivo dynamics of RSS, which is endogenously and ubiquitously produced in both prokaryotes and eukaryotes. RSS, an active sulfur molecule, maintains mitochondrial energy metabolism (Non-Patent Document 2), while exerting potent antioxidant (Non-Patent Document 1), anti-inflammatory (Non-Patent Document 3), and immunoregulatory functions (Non-Patent Document 3).

[0007] Meanwhile, there is an urgent need to clarify the pathogenesis of the novel coronavirus (SARS-CoV-2), which causes the novel coronavirus disease (COVID-19), and to establish preventive and therapeutic methods. SARS-CoV-2 protease inhibitors have been reported as therapeutic agents for COVID-19 (Non-Patent Documents 4, 5). [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Ida T, Sawa T, Ihara H, Tsuchiya Y, Watanabe Y, Kumagai Y, SuematsuM, Motohashi H, Fujii S, Matsunaga T, Yamamoto M, Ono K, Devarie-Baez NO, XianM, Fukuto JM, Akaike T. Reactive cysteine persulfides and S-polythiolationregulate oxidative stress and redox signaling. Proc Natl Acad Sci USA111:7606-7611 (2014). [Non-patent document 2] Akaike T, Ida T, Wei FY, Nishida M, Kumagai Y, Alam MM, Ihara H,Sawa T, Matsunaga T, Kasamatsu S, Nishimura A, Morita M, Tomizawa K, NishimuraA, Watanabe S, Inaba K, Shima H, Tanuma N, Jung M, Fujii S, Watanabe Y,Ohmuraya M, Nagy P, Feelisch M, Fukuto JM, Motohashi H. Cysteinyl-tRNAsynthetase governs cysteine polysulfidation and mitochondrial bioenergetics.Nat Commun 8:1177 (2017).

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0009] Based on the diverse physiological activities of active sulfur compounds present in living organisms, the inventors have developed an "antiviral drug containing an active sulfur compound as its main active ingredient," which inhibits viral proteases and has preventive and therapeutic effects against viral infections (Patent Application No. 2020-167343). The goal of this invention is to realize the usefulness of sulfur metabolites based on active sulfur metabolomics. [Means for solving the problem]

[0010] Based on active sulfur metabolomics, the present inventors investigated the usefulness of sulfur metabolites in relation to the antiviral effects of active sulfur compounds and found that sulfur metabolites function as biomarkers for infectious diseases. That is, the present invention is characterized by a biomarker for infectious diseases, which contains sulfur metabolites. The sulfur metabolites may be present in exhaled breath. The sulfur metabolites can be detected from exhaled breath condensate. The sulfur metabolites are particularly sulfite ions (HSO3 - ), thiosulfate ion (HS2O3 - ), and hydrogen disulfide ion (HS2 - ) or a salt thereof, or a compound, molecule, or derivative thereof containing these. Biomarkers for infectious diseases include biomarkers for diagnosing bacterial and viral infections, particularly novel coronavirus infection, biomarkers for diagnosing infectious pneumonia, particularly novel coronavirus-infected interstitial pneumonia and other pneumonias such as alveolar pneumonia, and biomarkers for diagnosing the progression of infection, particularly the risk of progression to severe novel coronavirus infection.

[0011] The present invention further provides a diagnostic system for detecting infection diagnostic biomarkers consisting of sulfur metabolites from exhaled breath, which includes an exhaled breath collector and exhaled breath collection device for collecting the exhaled breath of a subject, and an analytical device for qualitatively and quantitatively analyzing the sulfur metabolites in the collected exhaled breath, for determining infection, particularly a novel coronavirus infection diagnosis system. [Effects of the Invention]

[0012] According to the present invention, the usefulness of sulfur metabolites can be realized based on active sulfur metabolomics. [Brief explanation of the drawings]

[0013] [Figure 1] A list of 12 healthy subjects who are not infected with COVID-19 [Figure 2] List of 12 COVID-19 infected patients [Figure 3] Quantitative analysis results for healthy individuals and COVID-19 infected patients [Figure 4] Measurement results of sulfite ions (HSO3-) and thiosulfate ions (HS2O3-) in one COVID-19 patient when the condition worsened DETAILED DESCRIPTION OF THE INVENTION

[0014] We analyzed sulfur metabolites in the exhaled breath of 12 healthy individuals without COVID-19 infection (Figure 1) and 12 individuals infected with COVID-19 (inpatients at Tokai University Hospital, Figure 2). COVID-19 infection was determined based on PCR testing. Of the 12 individuals infected with COVID-19, two had mild symptoms and 10 had moderate symptoms, one of whom later developed severe symptoms. Classification of symptoms as mild, moderate, or severe was based on oxygen saturation, respiratory symptoms, and chest CT scans. This classification is described, for example, in the Novel Coronavirus Disease (COVID-19) Management Guide, Version 4.1.

[0015] Exhaled breath was collected using an exhaled breath collector (manufactured by GL Sciences Inc.) and an exhaled breath collection device (manufactured by GL Sciences Inc.) The exhaled breath collection device was pre-cooled to -20°C, and the subjects breathed through the exhaled breath collection device (mouthpiece type, mask type) for 5 to 10 minutes, which rapidly cooled the exhaled breath aerosol and collected approximately 1 ml of exhaled breath condensate.

[0016] The electrophilic alkylating agent, β-(4-hydroxyphenyl) ethyl iodoacetamide (HPE-IAM), was added to 25 μl of exhaled breath condensate at 5 mM, and the alkylation reaction was carried out at 37°C for 30 minutes. Formic acid was then added to 1% to stabilize the sulfur metabolites as HPE-IAM adducts. The electrophilic alkylating agent alkalizes the sulfur electron pair, inhibiting the decomposition of sulfur metabolites.

[0017] Furthermore, for each of the multiple sulfur metabolites, stable isotope-labeled HPE-IAM adduct standards were added to the solution to a concentration of 10 nM, and these were used as mass spectrometry samples. Sulfite ions (HSO3 - ), thiosulfate ion (HS2O3 - ), hydrogen disulfide ion (HS2 - The HPE-IAM adducts were quantitatively analyzed using a mass spectrometer. TM 35 μl of the solution was injected into a -8060 (Shimadzu) and quantitatively analyzed under the following MRM (multiple reaction monitoring) conditions (Table 1).

[0018] [Table 1]

[0019] Figure 3 shows the data from the quantitative analysis of these three ions for 12 healthy individuals and 12 COVID-19 patients. The concentrations of various sulfur metabolites in the breath condensate were generally at a lower level than in the body. This is thought to be due to factors such as the dilution of sulfur metabolites in the body in the breath and the inefficiency of breath collection. Furthermore, compared to healthy individuals, the sulfite ion (HSO3 - ), thiosulfate ion (HS2O3 - ), hydrogen disulfide ion (HS2 -) levels were found to be significantly higher.

[0020] The patient in Figure 2 (patient number 3) progressed from moderate to severe symptoms, but the sulfite ion (HSO3 - ), thiosulfate ion (HS2O3 - The measurement results of thiosulfate ion (HS2O3 - ), it was found that the production level was significantly higher in moderate and severe cases compared to healthy individuals. Later, in a moderate patient (patient number 3) whose pneumonia became severe, the sulfite ion (HSO3 - ) was found to increase significantly in patients with moderate disease, but no such increase was observed in patients who did not develop severe disease. - ) is a promising biomarker for assessing the risk of progression to severe pneumonia in COVID-19 patients.

[0021] Active sulfur has antioxidant activity, meaning that it is oxidized by oxidative stress and active oxygen, etc., so it can be said that the worsening of pneumonia, or in other words, the worsening of oxidative stress, causes the profile of sulfur metabolites to shift toward oxidation.

Claims

1. A biomarker for diagnosing a novel coronavirus infection, using a sulfur metabolite involved in the metabolism of an active sulfur compound in a living body, the sulfur metabolites are collected from exhaled breath; The sulfur metabolite is at least one of sulfite ion (HSO 3 − ), thiosulfate ion (HS 2 O 3 − ), and hydrogen disulfide ion (HS 2 − ). The biomarker.

2. The sulfur metabolite is used for diagnosing pneumonia caused by novel coronavirus infection. The biomarker of claim 1.

3. A biomarker described in claim 1 or claim 2, wherein the sulfite ion (HSO 3 − ) is used to assess the risk of a novel coronavirus infection becoming severe.

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