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1.
Mikrochim Acta ; 189(1): 34, 2021 12 23.
Article in English | MEDLINE | ID: covidwho-1633639

ABSTRACT

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings.


Subject(s)
Chemistry Techniques, Analytical/methods , DNA, Catalytic/chemistry , DNA, Complementary/analysis , Hydrogels/chemistry , RNA, Viral/genetics , SARS-CoV-2/chemistry , Capillary Action , Chemistry Techniques, Analytical/instrumentation , DNA, Catalytic/genetics , DNA, Complementary/genetics , Hydrogen-Ion Concentration , Inverted Repeat Sequences , Limit of Detection , Nucleic Acid Amplification Techniques , Nucleic Acid Hybridization
2.
Bull Exp Biol Med ; 172(3): 283-287, 2022 Jan.
Article in English | MEDLINE | ID: covidwho-1611428

ABSTRACT

We studied laboratory parameters of patients with COVID-19 against the background of chronic pathologies (cardiovascular pathologies, obesity, type 2 diabetes melitus, and cardiovascular pathologies with allergy to statins). A decrease in pH and a shift in the electrolyte balance of blood plasma were revealed in all studied groups and were most pronounced in patients with cardiovascular pathologies with allergy to statin. It was found that low pH promotes destruction of lipid components of the erythrocyte membranes in patients with chronic pathologies, which was seen from a decrease in Na+/K+-ATPase activity and significant hyponatrenemia. In patients with cardiovascular pathologies and allergy to statins, erythrocyte membranes were most sensitive to a decrease in pH, while erythrocyte membranes of obese patients showed the greatest resistance to low pH and oxidative stress.


Subject(s)
COVID-19/complications , Hyponatremia/etiology , Hypoxia/complications , Sodium-Potassium-Exchanging ATPase/physiology , Aged , COVID-19/metabolism , Cardiovascular Diseases/complications , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/virology , Case-Control Studies , Chronic Disease , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/virology , Drug Hypersensitivity/complications , Drug Hypersensitivity/metabolism , Drug Hypersensitivity/virology , Erythrocyte Membrane/metabolism , Erythrocytes/metabolism , Female , Fluid Shifts/physiology , Humans , Hydrogen-Ion Concentration , Hydroxymethylglutaryl-CoA Reductase Inhibitors/adverse effects , Hyponatremia/metabolism , Hyponatremia/virology , Hypoxia/metabolism , Lipid Peroxidation/physiology , Male , Middle Aged , Obesity/complications , Obesity/metabolism , Obesity/virology , Oxidative Stress/physiology , SARS-CoV-2/physiology , Sodium/metabolism , Stress, Physiological/physiology
3.
Drug Discov Ther ; 15(5): 268-272, 2021 Nov 21.
Article in English | MEDLINE | ID: covidwho-1542927

ABSTRACT

The inhibitory activity of electrolyzed reduced water (ERW) against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is the etiological agent responsible for coronavirus disease 2019 (COVID-19), was tested in vitro on Vero E6 cells using a plaque assay. Infectious virus titers of cells treated with ERW 100%, 50% and 33.3% solutions and phosphate buffered saline (PBS, negative control) and exposed to the virus suspension for 60 seconds were 2.25, 2.65, 3.21 and 7.38, respectively. ERW has a high pH and low surface tension. It is considered that the alkaline property of ERW breaks down phospholipids and proteins of envelopes. The role of pH and reducibility on the virucidal effect of ERW should be further evaluated. This study provides a foundation for utilizing ERW as an effective antiviral aqueous solution in a variety of applications.


Subject(s)
Antiviral Agents/pharmacology , SARS-CoV-2/drug effects , Water/pharmacology , Animals , Chlorocebus aethiops , Hydrogen-Ion Concentration , Vero Cells/virology , Viral Plaque Assay
4.
Sensors (Basel) ; 21(21)2021 Nov 01.
Article in English | MEDLINE | ID: covidwho-1512567

ABSTRACT

Potentiometric pH measurements have long been used for the bioanalysis of biofluids, tissues, and cells. A glass pH electrode and ion-sensitive field-effect transistor (ISFET) can measure the time course of pH changes in a microenvironment as a result of physiological and biological activities. However, the signal interpretation of passive pH sensing is difficult because many biological activities influence the spatiotemporal distribution of pH in the microenvironment. Moreover, time course measurement suffers from stability because of gradual drifts in signaling. To address these issues, an active method of pH sensing was developed for the analysis of the cell barrier in vitro. The microenvironmental pH is temporarily perturbed by introducing a low concentration of weak acid (NH4+) or base (CH3COO-) to cells cultured on the gate insulator of ISFET using a superfusion system. Considering the pH perturbation originates from the semi-permeability of lipid bilayer plasma membranes, induced proton dynamics are used for analyzing the biomembrane barriers against ions and hydrated species following interaction with exogenous reagents. The unique feature of the method is the sensitivity to the formation of transmembrane pores as small as a proton (H+), enabling the analysis of cell-nanomaterial interactions at the molecular level. The new modality of cell analysis using ISFET is expected to be applied to nanomedicine, drug screening, and tissue engineering.


Subject(s)
Biosensing Techniques , Transistors, Electronic , Electrodes , Hydrogen-Ion Concentration , Ions , Potentiometry
5.
Sci Rep ; 11(1): 21124, 2021 10 26.
Article in English | MEDLINE | ID: covidwho-1493211

ABSTRACT

Patients with coronavirus disease 2019 (COVID-19) can have increased risk of mortality shortly after intubation. The aim of this study is to develop a model using predictors of early mortality after intubation from COVID-19. A retrospective study of 1945 intubated patients with COVID-19 admitted to 12 Northwell hospitals in the greater New York City area was performed. Logistic regression model using backward selection was applied. This study evaluated predictors of 14-day mortality after intubation for COVID-19 patients. The predictors of mortality within 14 days after intubation included older age, history of chronic kidney disease, lower mean arterial pressure or increased dose of required vasopressors, higher urea nitrogen level, higher ferritin, higher oxygen index, and abnormal pH levels. We developed and externally validated an intubated COVID-19 predictive score (ICOP). The area under the receiver operating characteristic curve was 0.75 (95% CI 0.73-0.78) in the derivation cohort and 0.71 (95% CI 0.67-0.75) in the validation cohort; both were significantly greater than corresponding values for sequential organ failure assessment (SOFA) or CURB-65 scores. The externally validated predictive score may help clinicians estimate early mortality risk after intubation and provide guidance for deciding the most effective patient therapies.


Subject(s)
COVID-19/diagnosis , COVID-19/mortality , Intubation, Intratracheal/methods , Severity of Illness Index , Adolescent , Adult , Age Factors , Aged , Arterial Pressure , COVID-19/therapy , Female , Ferritins/blood , Humans , Hydrogen-Ion Concentration , Male , Middle Aged , New York , Nitrogen/metabolism , Oxygen/metabolism , Predictive Value of Tests , ROC Curve , Regression Analysis , Reproducibility of Results , Retrospective Studies , Sensitivity and Specificity , Vasoconstrictor Agents/pharmacology , Young Adult
6.
Sci Rep ; 10(1): 22200, 2020 12 17.
Article in English | MEDLINE | ID: covidwho-1493197

ABSTRACT

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is responsible for the novel coronavirus disease 2019 (COVID-19). An appealing antiviral drug target is the coronavirus 3C-like protease (3CLpro) that is responsible for the processing of the viral polyproteins and liberation of functional proteins essential for the maturation and infectivity of the virus. In this study, multiple thermal analytical techniques have been implemented to acquire the thermodynamic parameters of 3CLpro at different buffer conditions. 3CLpro exhibited relatively high thermodynamic stabilities over a wide pH range; however, the protease was found to be less stable in the presence of salts. Divalent metal cations reduced the thermodynamic stability of 3CLpro more than monovalent cations; however, altering the ionic strength of the buffer solution did not alter the stability of 3CLpro. Furthermore, the most stable thermal kinetic stability of 3CLpro was recorded at pH 7.5, with the highest enthalpy of activation calculated from the slope of Eyring plot. The biochemical and biophysical properties of 3CLpro explored here may improve the solubility and stability of 3CLpro for optimum conditions for the setup of an enzymatic assay for the screening of inhibitors to be used as lead candidates in the discovery of drugs and design of antiviral therapeutics against COVID-19.


Subject(s)
COVID-19/virology , Chymases/metabolism , Coronavirus 3C Proteases/metabolism , Peptide Hydrolases/metabolism , SARS-CoV-2/metabolism , Antiviral Agents/therapeutic use , Humans , Hydrogen-Ion Concentration , Protease Inhibitors/therapeutic use , Thermodynamics
7.
Molecules ; 26(19)2021 Oct 02.
Article in English | MEDLINE | ID: covidwho-1463766

ABSTRACT

Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-ß-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.


Subject(s)
Bacterial Proteins/metabolism , Chitin/metabolism , Chitinases/metabolism , Clostridium/metabolism , Bacterial Proteins/genetics , Catalytic Domain , Chitinases/chemistry , Chitinases/genetics , Clostridium/growth & development , Clostridium/isolation & purification , Gastrointestinal Microbiome , Humans , Hydrogen-Ion Concentration , Recombinant Proteins/genetics , Recombinant Proteins/metabolism
8.
Acta Crystallogr F Struct Biol Commun ; 77(Pt 10): 348-355, 2021 Oct 01.
Article in English | MEDLINE | ID: covidwho-1450488

ABSTRACT

Human coronavirus NL63 (HCoV-NL63), which belongs to the genus Alphacoronavirus, mainly infects children and the immunocompromized and is responsible for a series of clinical manifestations, including cough, fever, rhinorrhoea, bronchiolitis and croup. HCoV-NL63, which was first isolated from a seven-month-old child in 2004, has led to infections worldwide and accounts for 10% of all respiratory illnesses caused by etiological agents. However, effective antivirals against HCoV-NL63 infection are currently unavailable. The HCoV-NL63 main protease (Mpro), also called 3C-like protease (3CLpro), plays a vital role in mediating viral replication and transcription by catalyzing the cleavage of replicase polyproteins (pp1a and pp1ab) into functional subunits. Moreover, Mpro is highly conserved among all coronaviruses, thus making it a prominent drug target for antiviral therapy. Here, four crystal structures of HCoV-NL63 Mpro in the apo form at different pH values are reported at resolutions of up to 1.78 Å. Comparison with Mpro from other human betacoronaviruses such as SARS-CoV-2 and SARS-CoV reveals common and distinct structural features in different genera and extends knowledge of the diversity, function and evolution of coronaviruses.


Subject(s)
Coronavirus NL63, Human/chemistry , Crystallization/methods , Crystallography, X-Ray/methods , Humans , Hydrogen-Ion Concentration , Protein Conformation
9.
PLoS One ; 16(9): e0258018, 2021.
Article in English | MEDLINE | ID: covidwho-1443853

ABSTRACT

BACKGROUND: Data of critically ill COVID-19 patients are being evaluated worldwide, not only to understand the various aspects of the disease and to refine treatment strategies but also to improve clinical decision-making. For clinical decision-making in particular, prognostic factors of a lethal course of the disease would be highly relevant. METHODS: In this retrospective cohort study, we analyzed the first 59 adult critically ill Covid-19 patients treated in one of the intensive care units of the University Medical Center Regensburg, Germany. Using uni- and multivariable regression models, we extracted a set of parameters that allowed for prognosing in-hospital mortality. RESULTS: Within the cohort, 19 patients died (mortality 32.2%). Blood pH value, mean arterial pressure, base excess, troponin, and procalcitonin were identified as highly significant prognostic factors of in-hospital mortality. However, no significant differences were found for other parameters expected to be relevant prognostic factors, like low arterial partial pressure of oxygen or high lactate levels. In the multivariable logistic regression analysis, the pH value and the mean arterial pressure turned out to be the most influential prognostic factors for a lethal course.


Subject(s)
COVID-19/blood , COVID-19/mortality , Adult , Aged , Arterial Pressure/physiology , Blood Physiological Phenomena , Blood Pressure/physiology , Cohort Studies , Critical Illness/mortality , Female , Germany/epidemiology , Hospital Mortality/trends , Humans , Hydrogen-Ion Concentration , Intensive Care Units/trends , Male , Middle Aged , Mortality/trends , Prognosis , Retrospective Studies , Risk Factors , SARS-CoV-2/pathogenicity
10.
Ann Med ; 53(1): 1642-1645, 2021 12.
Article in English | MEDLINE | ID: covidwho-1404914

ABSTRACT

OBJECTIVE: To compare patients with DKA, hyperglycaemic hyperosmolar syndrome (HHS), or mixed DKA-HHS and COVID-19 [COVID (+)] to COVID-19-negative (-) [COVID (-)] patients with DKA/HHS from a low-income, racially/ethnically diverse catchment area. METHODS: A cross-sectional study was conducted with patients admitted to an urban academic medical center between 1 March and 30 July 2020. Eligible patients met lab criteria for either DKA or HHS. Mixed DKA-HHS was defined as meeting all criteria for either DKA or HHS with at least 1 criterion for the other diagnosis. RESULTS: A total of 82 participants were stratified by COVID-19 status and type of hyperglycaemic crisis [26 COVID (+) and 56 COVID (-)]. A majority were either Black or Hispanic. Compared with COVID (-) patients, COVID (+) patients were older, more Hispanic and more likely to have type 2 diabetes (T2D, 73% vs 48%, p < .01). COVID(+) patients had a higher mean pH (7.25 ± 0.10 vs 7.16 ± 0.16, p < .01) and lower anion gap (18.7 ± 5.7 vs 22.7 ± 6.9, p = .01) than COVID (-) patients. COVID (+) patients were given less intravenous fluids in the first 24 h (2.8 ± 1.9 vs 4.2 ± 2.4 L, p = .01) and were more likely to receive glucocorticoids (95% vs. 11%, p < .01). COVID (+) patients may have taken longer to resolve their hyperglycaemic crisis (53.3 ± 64.8 vs 28.8 ± 27.5 h, p = .09) and may have experienced more hypoglycaemia <3.9 mmol/L (35% vs 19%, p = .09). COVID (+) patients had a higher length of hospital stay (LOS, 14.8 ± 14.9 vs 6.5 ± 6.0 days, p = .01) and in-hospital mortality (27% vs 7%, p = .02). DISCUSSION: Compared with COVID (-) patients, COVID (+) patients with DKA/HHS are more likely to have T2D. Despite less severe metabolic acidosis, COVID (+) patients may require more time to resolve the hyperglycaemic crisis and experience more hypoglycaemia while suffering greater LOS and risk of mortality. Larger studies are needed to examine whether differences in management between COVID (+) and (-) patients affect outcomes with DKA/HHS.


Subject(s)
COVID-19/complications , Diabetic Ketoacidosis/complications , Hyperglycemic Hyperosmolar Nonketotic Coma/complications , Acid-Base Equilibrium , Adult , Age Factors , Aged , COVID-19/blood , COVID-19/epidemiology , COVID-19/therapy , Comorbidity , Cross-Sectional Studies , Diabetes Mellitus, Type 2/epidemiology , Diabetic Ketoacidosis/blood , Female , Fluid Therapy , Glucocorticoids/therapeutic use , Humans , Hydrogen-Ion Concentration , Hyperglycemic Hyperosmolar Nonketotic Coma/blood , Length of Stay , Male , Middle Aged , SARS-CoV-2 , Safety-net Providers
11.
J Biol Chem ; 297(4): 101175, 2021 10.
Article in English | MEDLINE | ID: covidwho-1401575

ABSTRACT

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.


Subject(s)
SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Amino Acid Motifs , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Humans , Hydrogen-Ion Concentration , Interferometry , Kinetics , Protein Binding , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , SARS-CoV-2/isolation & purification , Sequence Alignment , Spike Glycoprotein, Coronavirus/chemistry
12.
Trop Biomed ; 38(3): 343-352, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1404404

ABSTRACT

Outbreak of SARS-CoV-2 has been declared a pandemic, which is a serious threat to human health. The disease was named coronavirus disease 2019 (COVID-19). Until now, several vaccines and a few drugs have been approved for the prevention and treatment for COVID-19. Recently, the effect of some macrolides including clarithromycin (CAM) on COVID-19 has attracted attention. CAM is known to have diverse effects including immunomodulatory and immunosuppressive effects, autophagy inhibition, steroid sparing effect, reversibility of drug resistance, antineoplastic effect, antiviral effect as well as bacteriostatic/bactericidal effect. Many patients with COVID-19 died due to an overwhelming response of their own immune system characterized by the uncontrolled release of circulating inflammatory cytokines (cytokine release syndrome [CRS]). This CRS plays a major role in progressing pneumonia to acute respiratory distress syndrome (ARDS) in COVID-19 patients. It is noteworthy that CAM can suppress inflammatory cytokines responsible for CRS and also has anti-SARS-CoV-2 effect. Considering the rapidly progressive global disease burden of COVID 19, the application of CAM for treating COVID-19 needs to be urgently evaluated. Recently, an open-labeled non-randomized trial using CAM for treating COVID-19 (ACHIEVE) was initiated in Greece in May, 2020. Its results, though preprint, indicated that CAM treatment of patients with moderate COVID-19 was associated with early clinical improvement and containment of viral load. Thus, treatment with CAM as a single agent or combined with other anti-SARS CoV-2 drugs should be tried for treating COVID-19. In this article, we discussed the significance and usefulness of CAM in treating COVID-19.


Subject(s)
COVID-19/drug therapy , Clarithromycin/therapeutic use , Drug Repositioning , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/metabolism , Azithromycin/therapeutic use , Clarithromycin/pharmacology , Humans , Hydrogen-Ion Concentration , Immunologic Factors/pharmacology , SARS-CoV-2/drug effects
13.
Respir Care ; 66(1): 113-119, 2021 01.
Article in English | MEDLINE | ID: covidwho-1389654

ABSTRACT

BACKGROUND: Low airway surface pH is associated with many airway diseases, impairs antimicrobial host defense, and worsens airway inflammation. Inhaled Optate is designed to safely raise airway surface pH and is well tolerated in humans. Raising intracellular pH partially prevents activation of SARS-CoV-2 in primary normal human airway epithelial (NHAE) cells, decreasing viral replication by several mechanisms. METHODS: We grew primary NHAE cells from healthy subjects, infected them with SARS-CoV-2 (isolate USA-WA1/2020), and used clinical Optate at concentrations used in humans in vivo to determine whether Optate would prevent viral infection and replication. Cells were pretreated with Optate or placebo prior to infection (multiplicity of infection = 1), and viral replication was determined with plaque assay and nucleocapsid (N) protein levels. Healthy human subjects also inhaled Optate as part of a Phase 2a safety trial. RESULTS: Optate almost completely prevented viral replication at each time point between 24 h and 120 h, relative to placebo, on both plaque assay and N protein expression (P < .001). Mechanistically, Optate inhibited expression of major endosomal trafficking genes and raised NHAE intracellular pH. Optate had no effect on NHAE cell viability at any time point. Inhaled Optate was well tolerated in 10 normal subjects, with no change in lung function, vital signs, or oxygenation. CONCLUSIONS: Inhaled Optate may be well suited for a clinical trial in patients with pulmonary SARS-CoV-2 infection. However, it is vitally important for patient safety that formulations designed for inhalation with regard to pH, isotonicity, and osmolality be used. An inhalational treatment that safely prevents SARS-CoV-2 viral replication could be helpful for treating patients with pulmonary SARS-CoV-2 infection.


Subject(s)
Antiviral Agents/therapeutic use , COVID-19/drug therapy , Epithelial Cells/drug effects , Glycine/pharmacology , Isotonic Solutions/pharmacology , Lung/drug effects , SARS-CoV-2 , Virus Replication/drug effects , Administration, Inhalation , Antiviral Agents/administration & dosage , Cells, Cultured/drug effects , Glycine/administration & dosage , Healthy Volunteers , Humans , Hydrogen-Ion Concentration/drug effects , Isotonic Solutions/administration & dosage
14.
J Mol Biol ; 433(10): 166946, 2021 05 14.
Article in English | MEDLINE | ID: covidwho-1386061

ABSTRACT

Coronaviruses are a major infectious disease threat, and include the zoonotic-origin human pathogens SARS-CoV-2, SARS-CoV, and MERS-CoV (SARS-2, SARS-1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS-1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2' cleavage site is the bona fide SARS-1 FP. In this study, we used sequence alignment to identify the SARS-2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS-2 FP induces even greater membrane ordering than SARS-1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS-2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS-2 is greater. Both the membrane ordering of SARS-2 and SARS-1 FPs are dependent on Ca2+, but that of SARS-2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS-1 FP, but the two Ca2+ binding sites of SARS-2 exhibit greater cooperativity. This Ca2+ dependence by the SARS-2 FP is very ion-specific. These results show that Ca2+ is an important regulator that interacts with the SARS-2 FP and thus plays a significant role in SARS-2 viral entry. This could lead to therapeutic solutions that either target the FP-calcium interaction or block the Ca2+ channel.


Subject(s)
Calcium/metabolism , Cell Membrane/metabolism , SARS Virus/metabolism , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Viral Fusion Proteins/metabolism , Amino Acid Sequence , Binding Sites , Calcium/pharmacology , Calorimetry , Cell Membrane/drug effects , Cell Membrane/virology , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , SARS Virus/drug effects , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Thermodynamics , Viral Fusion Proteins/chemistry , Viral Fusion Proteins/genetics , Virus Internalization/drug effects
15.
J Physiol ; 599(11): 2785-2786, 2021 06.
Article in English | MEDLINE | ID: covidwho-1388428
16.
J Phys Chem Lett ; 12(26): 6218-6226, 2021 Jul 08.
Article in English | MEDLINE | ID: covidwho-1387122

ABSTRACT

Following our previous work ( Chem. Sci. 2021, 12, 4889-4907), we study the structural dynamics of the SARS-CoV-2 Main Protease dimerization interface (apo dimer) by means of microsecond adaptive sampling molecular dynamics simulations (50 µs) using the AMOEBA polarizable force field (PFF). This interface is structured by a complex H-bond network that is stable only at physiological pH. Structural correlations analysis between its residues and the catalytic site confirms the presence of a buried allosteric site. However, noticeable differences in allosteric connectivity are observed between PFFs and non-PFFs. Interfacial polarizable water molecules are shown to appear at the heart of this discrepancy because they are connected to the global interface H-bond network and able to adapt their dipole moment (and dynamics) to their diverse local physicochemical microenvironments. The water-interface many-body interactions appear to drive the interface volume fluctuations and to therefore mediate the allosteric interactions with the catalytic cavity.


Subject(s)
Molecular Dynamics Simulation , SARS-CoV-2/metabolism , Viral Matrix Proteins/chemistry , Water/chemistry , Allosteric Site , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Dimerization , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , SARS-CoV-2/isolation & purification , Viral Matrix Proteins/metabolism
17.
Nat Struct Mol Biol ; 27(12): 1202-1208, 2020 12.
Article in English | MEDLINE | ID: covidwho-1387444

ABSTRACT

An essential protein of the SARS-CoV-2 virus, the envelope protein E, forms a homopentameric cation channel that is important for virus pathogenicity. Here we report a 2.1-Å structure and the drug-binding site of E's transmembrane domain (ETM), determined using solid-state NMR spectroscopy. In lipid bilayers that mimic the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) membrane, ETM forms a five-helix bundle surrounding a narrow pore. The protein deviates from the ideal α-helical geometry due to three phenylalanine residues, which stack within each helix and between helices. Together with valine and leucine interdigitation, these cause a dehydrated pore compared with the viroporins of influenza viruses and HIV. Hexamethylene amiloride binds the polar amino-terminal lumen, whereas acidic pH affects the carboxy-terminal conformation. Thus, the N- and C-terminal halves of this bipartite channel may interact with other viral and host proteins semi-independently. The structure sets the stage for designing E inhibitors as antiviral drugs.


Subject(s)
Coronavirus Envelope Proteins/chemistry , Lipid Bilayers/chemistry , SARS-CoV-2/chemistry , Amantadine/chemistry , Amiloride/analogs & derivatives , Amiloride/chemistry , Antiviral Agents/chemistry , Coronavirus Envelope Proteins/genetics , Dimyristoylphosphatidylcholine/chemistry , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Models, Molecular , Phenylalanine/chemistry , Phospholipids/chemistry , Protein Conformation , Protein Domains , SARS-CoV-2/genetics
18.
Cell Host Microbe ; 28(6): 867-879.e5, 2020 12 09.
Article in English | MEDLINE | ID: covidwho-1385264

ABSTRACT

The SARS-CoV-2 spike employs mobile receptor-binding domains (RBDs) to engage the human ACE2 receptor and to facilitate virus entry, which can occur through low-pH-endosomal pathways. To understand how ACE2 binding and low pH affect spike conformation, we determined cryo-electron microscopy structures-at serological and endosomal pH-delineating spike recognition of up to three ACE2 molecules. RBDs freely adopted "up" conformations required for ACE2 interaction, primarily through RBD movement combined with smaller alterations in neighboring domains. In the absence of ACE2, single-RBD-up conformations dominated at pH 5.5, resolving into a solitary all-down conformation at lower pH. Notably, a pH-dependent refolding region (residues 824-858) at the spike-interdomain interface displayed dramatic structural rearrangements and mediated RBD positioning through coordinated movements of the entire trimer apex. These structures provide a foundation for understanding prefusion-spike mechanics governing endosomal entry; we suggest that the low pH all-down conformation potentially facilitates immune evasion from RBD-up binding antibody.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Pandemics , Spike Glycoprotein, Coronavirus/ultrastructure , Amino Acid Sequence/genetics , Angiotensin-Converting Enzyme 2/ultrastructure , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19/pathology , COVID-19/virology , Cryoelectron Microscopy , Endosomes/ultrastructure , Humans , Hydrogen-Ion Concentration , Protein Binding , Protein Domains , Receptors, Virus/genetics , Receptors, Virus/ultrastructure , SARS-CoV-2/genetics , SARS-CoV-2/ultrastructure , Spike Glycoprotein, Coronavirus/genetics
19.
Angew Chem Int Ed Engl ; 59(45): 20154-20160, 2020 11 02.
Article in English | MEDLINE | ID: covidwho-1384106

ABSTRACT

Phosphoramidates composed of an amino acid and a nucleotide analogue are critical metabolites of prodrugs, such as remdesivir. Hydrolysis of the phosphoramidate liberates the nucleotide, which can then be phosphorylated to become the pharmacologically active triphosphate. Enzymatic hydrolysis has been demonstrated, but a spontaneous chemical process may also occur. We measured the rate of enzyme-free hydrolysis for 17 phosphoramidates of ribonucleotides with amino acids or related compounds at pH 7.5. Phosphoramidates of proline hydrolyzed fast, with a half-life time as short as 2.4 h for Pro-AMP in ethylimidazole-containing buffer at 37 °C; 45-fold faster than Ala-AMP and 120-fold faster than Phe-AMP. Crystal structures of Gly-AMP, Pro-AMP, ßPro-AMP and Phe-AMP bound to RNase A as crystallization chaperone showed how well the carboxylate is poised to attack the phosphoramidate, helping to explain this reactivity. Our results are significant for the design of new antiviral prodrugs.


Subject(s)
Amides/metabolism , Amino Acids/chemistry , Nucleotides/metabolism , Phosphoric Acids/metabolism , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/metabolism , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/metabolism , Amides/chemistry , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/pathology , COVID-19/virology , Catalytic Domain , Crystallography, X-Ray , Half-Life , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Dynamics Simulation , Nucleotides/chemistry , Phosphoric Acids/chemistry , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/isolation & purification
20.
Cell Res ; 31(10): 1047-1060, 2021 10.
Article in English | MEDLINE | ID: covidwho-1380899

ABSTRACT

The outbreak of SARS-CoV-2 (SARS2) has caused a global COVID-19 pandemic. The spike protein of SARS2 (SARS2-S) recognizes host receptors, including ACE2, to initiate viral entry in a complex biomechanical environment. Here, we reveal that tensile force, generated by bending of the host cell membrane, strengthens spike recognition of ACE2 and accelerates the detachment of spike's S1 subunit from the S2 subunit to rapidly prime the viral fusion machinery. Mechanistically, such mechano-activation is fulfilled by force-induced opening and rotation of spike's receptor-binding domain to prolong the bond lifetime of spike/ACE2 binding, up to 4 times longer than that of SARS-S binding with ACE2 under 10 pN force application, and subsequently by force-accelerated S1/S2 detachment which is up to ~103 times faster than that in the no-force condition. Interestingly, the SARS2-S D614G mutant, a more infectious variant, shows 3-time stronger force-dependent ACE2 binding and 35-time faster force-induced S1/S2 detachment. We also reveal that an anti-S1/S2 non-RBD-blocking antibody that was derived from convalescent COVID-19 patients with potent neutralizing capability can reduce S1/S2 detachment by 3 × 106 times under force. Our study sheds light on the mechano-chemistry of spike activation and on developing a non-RBD-blocking but S1/S2-locking therapeutic strategy to prevent SARS2 invasion.


Subject(s)
COVID-19/diagnosis , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Tensile Strength , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/immunology , Binding Sites , COVID-19/therapy , COVID-19/virology , Humans , Hydrogen-Ion Concentration , Immunization, Passive , Molecular Dynamics Simulation , Protein Binding , Protein Domains/immunology , Protein Subunits/chemistry , Protein Subunits/immunology , Protein Subunits/metabolism , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Virus Internalization
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