Increasing the Level of Neopterin in patients infected with SARS-CoV-2 Indicates Fatal Mortality

Background: The severity of Coronavirus Disease-2019 (COVID-19) cases is associated with hyperinflammation. Patients with critical and severe COVID-19 have been observed to have high amounts of circulating cytokines. Neopterin, a crucial cytokine in the antiviral immune response that is released by macrophages upon stimulation with interferon-gamma, can be utilized to forecast the severity of illness in COVID-19 patients. Methods: The study included 185 patients with COVID-19. The patients with COVID-19 were divided into three groups according to disease severity as critical disease (n=51), severe disease (n=81), and moderate disease (n=53). All basic demographic and clinical data of the patients were recorded and blood samples were collected. Results: Neopterin levels were significantly higher in critical COVID-19 patients compared with severe and moderate COVID-19 patients (p < 0.0001). Further, neopterin showed significantly higher levels in the age group >50 years of patients with COVID-19 than in the age group <50 years. Neopterin levels were correlated with WBCs, Platelet, CRP, D-Dimer, Ferritin, Fibrinogen, IL-6, and Procalcitonin levels positively (ρ= 0.569, 0.474, 0.338, 0.696, 0.605, 0.77, 0.727, and 0.585;p < 0.01 respectively), and correlated with BMI, SpO2, and lymphocyte negatively (ρ= - 0.165;p < 0.05, p= - 0.754, - 0. 548;p < 0.01 respectively). A cutoff value of 23.62 nmol/L for neopterin predicted COVID-19 with a sensitivity of 95.7% and a specificity of 95.5% (AUC: 0.986;p < 0.0001). Conclusion: Neopterin may be a useful prognostic biomarker for assessing the severity of COVID-19.

A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors.

The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC50 value of 24.26 ± 0.4 µM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening.

Identification of potent inhibitors of SARS-CoV-2 exoribonuclease by fluorescence polarization assay

The emergence of SARS-CoV-2 has triggered a pandemic with devastating consequences to the world. One of the proteins essential to the virus life cycle is nsp14, which is a bifunctional protein that encodes a 3'to 5' exoribonuclease activity in its N-terminus, and a methyl transferase activity in its C-terminus. Nsp14 in complex with the accessory protein nsp10 is involved in a proofreading mechanism that ensures the genetic stability of its massive viral genome, and is associated to the resistance against nucleotide analogs targeting the polymerase nsp12. Because of its key role, nsp14-nsp10 complex constitutes an attractive target for antiviral development. Here we present a fluorescence polarization (FP) assay development to measure the exoribonuclease activity and its inhibition in vitro. The FP method is sensitive, robust, amenable to miniaturization and offers confirmation by visualizing the degradation of the fluorescent RNA in acrylamide gels. We performed a screening of a focused library of 113 metal chelators at 20 and 5 μM compound concentration and IC50 measurement of 9 hits showing efficiency at micromolar level. We also tested the focused library in SARS-CoV-2 infected Vero cells and we confirmed 3 hits previously detected in the in vitro screening out of 6 promising inhibitors. In conclusion the FP method proposed is a reliable tool to discover inhibitors of the SARS-CoV-2 exoribonuclease activity and will help to find new antivirals to be used in combination with nucleoside analogs.

[Identifying SARS-CoV-2 main protease inhibitors by a novel sandwich-like fluorescence polarization screening assay].

SARS-CoV-2 main protease (Mpro) is responsible for polyprotein cleavage to release non-structural proteins (nsps) for viral genomic RNA replication, and its homologues are absent in human cells. Therefore, Mpro has been regarded as one of the ideal drug targets for the treatment of coronavirus disease 2019 (COVID-19). In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel sandwich-like FP screening assay for quick discovery of SARS-CoV-2 Mpro inhibitors from a natural product library. With this screening assay, anacardic acid (AA) and 1, 2, 3, 4, 6-O-pentagalloylglucose (PGG) were found to be the competitive inhibitor and mixed-type inhibitor targeting Mpro, respectively. Importantly, our results showed that the majority of the reported Mpro inhibitors are promiscuous cysteine inhibitors that are not specific to Mpro. In summary, this novel sandwich-like FP screening assay is simple, sensitive, and robust, which is ideal for large-scale screening. Natural products AA and PGG will be the promising lead compounds for generating more potent antiviral agents targeting Mpro, and the stringent hit validation at the early stage of drug discovery is urgently needed.

Identification of potent inhibitors of arenavirus and SARS-CoV-2 exoribonucleases by fluorescence polarization assay.

Viral exoribonucleases are uncommon in the world of RNA viruses. To date, they have only been identified in the Arenaviridae and the Coronaviridae families. The exoribonucleases of these viruses play a crucial role in the pathogenicity and interplay with host innate immune response. Moreover, coronaviruses exoribonuclease is also involved in a proofreading mechanism ensuring the genetic stability of the viral genome. Because of their key roles in virus life cycle, they constitute attractive target for drug design. Here we developed a sensitive, robust and reliable fluorescence polarization assay to measure the exoribonuclease activity and its inhibition in vitro. The effectiveness of the method was validated on three different viral exoribonucleases, including SARS-CoV-2, Lymphocytic Choriomeningitis and Machupo viruses. We performed a screening of a focused library consisting of 113 metal chelators. Hit compounds were recovered with an IC50 at micromolar level. We confirmed 3 hits in SARS-CoV-2 infected Vero-E6 cells.

Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. RESULTS: In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. CONCLUSIONS: This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

Facile and rapid detection of SARS-CoV-2 antibody based on a noncompetitive fluorescence polarization immunoassay in human serum samples.

Antibody detection methods for viral infections have received broad attention due to the COVID-19 pandemic. In addition, there remains an ever-increasing need to quantitatively evaluate the immune response to develop vaccines and treatments for COVID-19. Here, we report an analytical method for the rapid and quantitative detection of SARS-CoV-2 antibody in human serum by fluorescence polarization immunoassay (FPIA). A recombinant SARS-CoV-2 receptor binding domain (RBD) protein labeled with HiLyte Fluor 647 (F-RBD) was prepared and used for FPIA. When the anti-RBD antibody in human serum binds to F-RBD, the degree of polarization (P) increases by suppressing the rotational diffusion of F-RBD. The measurement procedure required only mixing a reagent containing F-RBD with serum sample and measuring the P value with a portable fluorescence polarization analyzer after 15 min incubation. We evaluated analytical performance of the developed FPIA system using 30 samples: 20 COVID-19 positive sera and 10 negative sera. The receiver operating characteristic curve drawn with the obtained results showed that this FPIA system had high accuracy for discriminating COVID-19 positive or negative serum (AUC = 0.965). The total measurement time was about 20 min, and the serum volume required for measurement was 0.25 µL. Therefore, we successfully developed the FPIA system that enables rapid and easy quantification of SARS-CoV-2 antibody. It is believed that our FPIA system will facilitate rapid on-site identification of infected persons and deepen understanding of the immune response to COVID-19.

Fluorescence polarization system for rapid COVID-19 diagnosis.

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.

Rapid detection of anti-H5 avian influenza virus antibody by fluorescence polarization immunoassay using a portable fluorescence polarization analyzer.

A rapid, facile and selective detection of anti-H5 subtype avian influenza virus (AIV) antibody in serum by fluorescence polarization immunoassay (FPIA) was achieved. A fragment of recombinant H5 subtype AIV hemagglutinin was produced and labeled with fluorescein to use it as a labeled antigen in FPIA. This labeled antigen was mixed with anti-AIV sera (H1-H16 subtypes) and FP of the mixture was measured using a portable FP analyzer on a microdevice. It was found that FP increased in proportion to the concentration of anti-H5 AIV antibody (serum) and was significantly higher than FP obtained with the other sera. The selective detection of anti-H5 subtype AIV antibody was confirmed. The required volume of original sample was 2 µL and analysis time was within 20 min. This detection system realizes an efficient on-site diagnosis and surveillance of AIV.