scholarly journals The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 425 ◽  
Author(s):  
Javed Akhter ◽  
Grégory Quéromès ◽  
Krishna Pillai ◽  
Vahan Kepenekian ◽  
Samina Badar ◽  
...  
Keyword(s):  
Envelope Protein ◽  
Disulfide Bonds ◽  
Host Cells ◽  
Nasal Administration ◽  
Disulfide Bridges ◽  
Dependent Inactivation ◽  
Virus Culture ◽  
Protein Disulfide Bonds ◽  
Protein Disulfide

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

scholarly journals The combination of Bromelain and Acetylcysteine (BromAc) synergistically inactivates SARS-CoV-2

2020 ◽  
Author(s):  
Javed Akhter ◽  
Grégory Quéromès ◽  
Krishna Pillai ◽  
Vahan Kepenekian ◽  
Samina Badar ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Single Agent ◽  
Drug Repurposing ◽  
Envelope Proteins ◽  
Host Cells ◽  
Nasal Administration ◽  
Replication Capacity ◽  
Wild Type ◽  
Virus Culture

AbstractBackground and objectivesSARS-CoV-2 infection is the cause of a worldwide pandemic, currently with limited therapeutic options. Whilst vaccines are at the forefront of the therapeutic initiative, drug repurposing remains a promising approach for SARS-CoV-2 treatment. BromAc (Bromelain & Acetylcysteine) has synergistic action against glycoproteins by the synchronous breakage of glycosidic linkages and disulfide bonds. The spike protein of SARS-CoV-2, formed of glycoprotein and disulfide bridges for stabilization, represents an attractive target as it is essential for binding to the ACE2 receptor in host cells present in nasal mucosa. We sought to determine the effect of BromAc on the Spike and Envelope proteins and its potential to reduce infectivity in host cells.DesignRecombinant Spike and Envelope proteins were treated by single agent and combination BromAc at 50 and 100 µg/20mg/mL and analyzed by electrophoresis. Ultraviolet analysis of disulfide bond reduction was performed for both Spike and Envelope proteins after treatment with Acetylcysteine. In vitro whole virus culture inactivation of pre-treated wild type and an S1/S2 Spike mutant SARS-CoV-2 with BromAc from 25 to 250 µg/20mg/mL was measured by cytopathic effect, cell lysis assay, and replication capacity by RT-PCR.ResultsRecombinant Spike and Envelope SARS-CoV-2 proteins were fragmented by BromAc at both 50 and 100 µg/20mg/mL whilst single agents had minimal effect. Spike and Envelope protein disulfide bonds were reduced by Acetylcysteine. In vitro whole virus culture of both wild type and Spike mutant SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents.ConclusionBromAc disintegrates SARS-CoV-2 Spike and Envelope proteins. In vitro tests on whole virus support this finding with inactivation of its replication capacity most strongly at 100 and 250 µg/20mg/mL BromAc, even in Spike mutant virus. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.Author SummaryThere is currently no suitable therapeutic treatment for early SARS-CoV-2 aimed to prevent disease progression. BromAc is under clinical development by the authors for mucinous cancers due to its ability to alter complex glycoproteins structure. The potential of BromAc on SARS-CoV-2 Spike and Envelope glycoproteins stabilized by disulfide bonds was examined and found to disintegrate recombinant Spike and Envelope proteins whilst reducing disulfide stabilizer bridges. BromAc also showed an inhibitory effect on wild-type and Spike mutant SARS-CoV-2 by inactivation of its replication capacity in vitro. Hence, BromAc may be an effective therapeutic agent for early SARS-CoV-2 infection, despite mutations, and even have potential as a prophylactic in people at high risk of infection.

scholarly journals Surface Accessibility of the 70-Kilodalton Chlamydia trachomatis Heat Shock Protein following Reduction of Outer Membrane Protein Disulfide Bonds

2002 ◽  
Vol 70 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Jane E. Raulston ◽  
Carolyn H. Davis ◽  
Terry R. Paul ◽  
J. Dave Hobbs ◽  
Priscilla B. Wyrick
Keyword(s):  
Heat Shock ◽  
Chlamydia Trachomatis ◽  
Outer Membrane ◽  
Disulfide Bonds ◽  
Outer Membrane Proteins ◽  
Host Cells ◽  
Productive Infection ◽  
Protein Disulfide Bonds ◽  
Protein Disulfide ◽  
Substrate Binding Domain

ABSTRACT Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is the Chlamydia trachomatis Hsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5′-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.

scholarly journals Stabilization of phage T4 lysozyme by engineered disulfide bonds

1989 ◽  
Vol 86 (17) ◽  
pp. 6562-6566 ◽  
Author(s):  
M Matsumura ◽  
W J Becktel ◽  
M Levitt ◽  
B W Matthews
Keyword(s):  
Disulfide Bonds ◽  
Theoretical Calculations ◽  
Equilibrium Constants ◽  
T4 Lysozyme ◽  
Disulfide Bridges ◽  
Large Loop ◽  
Phage T4 ◽  
Flexible Part ◽  
Free Phage

Four different disulfide bridges (linking positions 9-164, 21-142, 90-122, and 127-154) were introduced into a cysteine-free phage T4 lysozyme at sites suggested by theoretical calculations and computer modeling. The new cysteines spontaneously formed disulfide bonds on exposure to air in vitro. In all cases the oxidized (crosslinked) lysozyme was more stable than the corresponding reduced (noncrosslinked) enzyme toward thermal denaturation. Relative to wild-type lysozyme, the melting temperatures of the 9-164 and 21-142 disulfide mutants were increased by 6.4 degrees C and 11.0 degrees C, whereas the other two mutants were either less stable or equally stable. Measurement of the equilibrium constants for the reduction of the engineered disulfide bonds by dithiothreitol indicates that the less thermostable mutants tend to have a less favorable crosslink in the native structure. The two disulfide bridges that are most effective in increasing the stability of T4 lysozyme have, in common, a large loop size and a location that includes a flexible part of the molecule. The results suggest that stabilization due to the effect of the crosslink on the entropy of the unfolded polypeptide is offset by the strain energy associated with formation of the disulfide bond in the folded protein. The design of disulfide bridges is discussed in terms of protein flexibility.

scholarly journals Reduction of protein disulfide bonds in an oxidizing environment

FEBS Letters ◽  
1997 ◽  
Vol 401 (2-3) ◽  
pp. 104-108 ◽  
Author(s):  
Irina Majoul ◽  
David Ferrari ◽  
Hans-Dieter Söling
Keyword(s):  
Disulfide Bonds ◽  
Protein Disulfide Bonds ◽  
Protein Disulfide

Rapid and Sensitive Determination of Protein Disulfide Bonds

Proteins ◽  
1987 ◽  
pp. 493-501 ◽  
Author(s):  
Hsieng S. Lu ◽  
Michael L. Klein ◽  
Richard R. Everett ◽  
Por-Hsiung Lai
Keyword(s):  
Disulfide Bonds ◽  
Sensitive Determination ◽  
Protein Disulfide Bonds ◽  
Protein Disulfide

scholarly journals Production of In Vitro Lytic Characteristics of Paroxysmal Nocturnal Hemoglobinuria Erythrocytes in Normal Erythrocytes

Blood ◽  
1968 ◽  
Vol 32 (1) ◽  
pp. 49-58 ◽  
Author(s):  
HERBERT E. KANN ◽  
CHARLES E. MENGEL ◽  
WILHELM D. MERIWETHER ◽  
LARRY EBBERT
Keyword(s):  
Disulfide Bonds ◽  
Reduced Glutathione ◽  
Lipid Peroxide ◽  
Acetylcholinesterase Activity ◽  
Membrane Lipid ◽  
Alkaline Solutions ◽  
Magnesium Ion ◽  
Ph Optimum ◽  
Protein Disulfide Bonds

Abstract The concept that production of a "perfect" PNH RBC, artificially, might supply information as to the nature of the defect(s) in PNH RBCs was the basis for a study in which normal RBCs were studied after preincubation in concentrated, alkaline solutions of reduced glutathione. These RBCs exhibited the following features of PNH RBCs. 1. Sensitivity to lysis by acidified serum a. pH optimum identical to that of PNH RBCs b. complete prevention by prior heating of serum to 56° C for 30 minutes c. complete prevention by addition of dextran to serum d. complete prevention by removal of magnesium ion from serum, reversed by re-addition of magnesium ion to serum 2. Positive thrombin lysis test. 3. Positive sucrose lysis test. 4. No agglutination in type-compatible serum. 5. No greater than normal agglutination in serum containing isoantibodies or elevated titers of cold agglutinins, but marked enhancement of lytic sensitivity to these antibodies, identical to that achieved with "natural" PNH cells. 6. Positive Hegglin-Maier test. 7. Decreased acetylcholinesterase activity. 8. Increased lysis and lipid peroxide formation during incubation with hydrogen peroxide. The broad scope of these similarities permits cautious speculation that some biochemical feature(s) of PNH RBCs may have been produced in normal RBCs, artificially. The mechanism by which reduced glutathione produces the change is uncertain, but may involve either oxidation of membrane lipid or splitting of membrane protein disulfide bonds, or both.

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