It is an urgent demand worldwide to control the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) are key targets to discover SARS-CoV-2 inhibitors. After screening 12 Chinese herbal medicines and 125 compounds from licorice, we found that a popular natural product schaftoside inhibited 3CLpro and PLpro with IC50 values of 1.73 ± 0.22 and 3.91 ± 0.19 μmol/L, respectively, and inhibited SARS-CoV-2 virus in Vero E6 cells with EC50 of 11.83 ± 3.23 μmol/L. Hydrogen–deuterium exchange mass spectrometry analysis, quantum mechanics/molecular mechanics calculations, together with site-directed mutagenesis indicated the antiviral activities of schaftoside were related with non-covalent interactions with H41, G143 and R188 of 3CLpro, and K157, E167 and A246 of PLpro. Moreover, proteomics analysis and cytokine assay revealed that schaftoside also regulated immune response and inflammation of the host cells. The anti-inflammatory activities of schaftoside were confirmed on lipopolysaccharide-induced acute lung injury mice. Schaftoside showed good safety and pharmacokinetic property, and could be a promising drug candidate for the prevention and treatment of COVID-19.
Currently, human health due to corona virus disease 2019 (COVID-19) pandemic has been seriously threatened. The coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19. However, the efficacy is compromised by the SARS-CoV-2 evolvement and mutation. Here we report the SARS-CoV-2 S protein receptor-binding domain (RBD) inhibitor licorice-saponin A3 (A3) could widely inhibit RBD of SARS-CoV-2 variants, including Beta, Delta, and Omicron BA.1, XBB and BQ1.1. Furthermore, A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells, with EC50 of 1.016 μM. The mechanism was related with binding with Y453 of RBD determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis combined with quantum mechanics/molecular mechanics (QM/MM) simulations. Interestingly, phosphoproteomics analysis and multi fluorescent immunohistochemistry (mIHC) respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 MAPK pathways and rebalancing the corresponding immune dysregulation. This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.
Wound infections are prone to attacks from infectious pathogens, including multidrug resistant bacteria that render conventional antimicrobials ineffective. Recently, lysins have been proposed as alternatives to conventional antimicrobials to tackle the menace of multidrug resistance pathogens. The coupling of lysins with a material that will cover the wound may prove beneficial in both protecting and treating wound infections. Hence, in this study, a Gram-negative lysin, LysP53, was coupled with a thermosensitive hydrogel, poloxamer P407, and its efficacy to treat wound infection was tested. In vitro, the addition of LysP53 to the poloxamer did not affect its thermosensitive characteristics, nor did it affect the hydrogel structure. Moreover, the lysin hydrogel could hydrolyze the peptidoglycan, demonstrating that it may have bactericidal activity. Up to 10.4% of LysP53 was released from the hydrogel gradually within 24 h, which led to a 4-log reduction of stationary phase
Bacteriophages exert strong selection on their bacterial hosts to evolve resistance. At the same time, the fitness costs on bacteria following phage resistance may change their virulence, which may affect the therapeutic outcomes of phage therapy. In this study, we set out to assess the costs of phage resistance on the in vitro virulence of priority 1 nosocomial pathogenic bacterium, Acinetobacter baumannii. By subjecting phage-resistant variant Ev5-WHG of A. baumannii WHG40004 to several in vitro virulence profiles, we found that its resistance to phage is associated with reduced fitness in host microenvironments. Also, the mutant exhibited impaired adhesion and invasion to mammalian cells, as well as increased susceptibility to macrophage phagocytosis. Furthermore, the whole-genome sequencing of the mutant revealed that there exist multiple mutations which may play a role in phage resistance and altered virulence. Altogether, this study demonstrates that resistance to phage can significantly alter phenotypes associated with virulence in Acinetobacter baumannii.
Phage treatment of bacterial infections has offered some hope even as the crisis of antimicrobial resistance continues to be on the rise. However, bacterial resistance to phage is another looming challenge capable of undermining the effectiveness of phage therapy. Moreover, the consideration of including phage therapy in modern medicine calls for more careful research around every aspect of phage study. In an attempt to adequately prepare for the events of phage resistance, many studies have attempted to experimentally evolve phage resistance in different bacterial strains, as well as train phages to evolve counter-infectivity of resistant bacterial mutants, in view of answering such questions as coevolutionary dynamics between phage and bacteria, mechanisms of phage resistance, fitness costs of phage resistance on bacteria, etc. In this review, we summarised many such studies and by careful examination, highlighted critical issues to the outcome of phage therapy. We also discuss the insufficiency of many of these in vitro studies to represent actual disease conditions during phage application, alongside other complications that exist in phage-bacterial evolutionary interactions. Conclusively, we present the exploitation of phage-bacterial interactions for successful infection managements, as well as some future perspectives to direct phage research.
The evolution of SARS-CoV-2 virus has resulted in the global pandemic COVID-19. Given the advent of subvariants, it is urgent to develop novel drugs. This work aims to discover SARS-CoV-2 inhibitors from Scutellaria baicalensis Georgi targeting the proteases 3CLpro and PLpro. After screening 25 flavonoids, we revealed that chrysin 7-O-β-D-glucuronide could potently inhibit SARS-CoV-2 on Vero E6 cells, with EC50 of 8.72 μM. Surface plasmon resonance, site-directed mutagenesis and enzymatic activity measurements indicated chrysin-7-O-β-D-glucuronide inhibits SARS-CoV-2 through binding to H41 of 3CLpro, and K157 and E167 of PLpro, and hydrogen-deuterium exchange mass spectrometry analysis showed PLpro conformation has remarkable changes caused by chrysin-7-O-β-D-glucuronide. Finally, we revealed anti-inflammatory activity of chrysin 7-O-β-D-glucuronide mainly caused by decreasing level of proinflammatory cytokines IL-1β and IL-6.
The COVID-19 global epidemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a great public health emergency. Discovering antiviral drug candidates is urgent for the prevention and treatment of COVID-19. This work aims to discover natural SARS-CoV-2 inhibitors from the traditional Chinese herbal medicine licorice. We screened 125 small molecules from Glycyrrhiza uralensis Fisch. (licorice, Gan-Cao) by virtual ligand screening targeting the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. Potential hit compounds were further evaluated by ELISA, SPR, luciferase assay, antiviral assay and pharmacokinetic study. The triterpenoids licorice-saponin A3 (A3) and glycyrrhetinic acid (GA) could potently inhibit SARS-CoV-2 infection, with EC50 of 75 nM and 3.17 µM, respectively. Moreover, we reveal that A3 mainly targets the nsp7 protein, and GA binds to the spike protein RBD of SARS-CoV-2. In this work, we found GA and A3 from licorice potently inhibit SARS-CoV-2 infection by affecting entry and replication of the virus. Our findings indicate that these triterpenoids may contribute to the clinical efficacy of licorice for COVID-19 and could be promising candidates for antiviral drug development.
Abstract Objectives Calcium-binding motifs are shared by multiple bacteriophage lysins; however, the influence of calcium on the enzymatic activity and host range of these enzymes is still not understood. To address this, ClyF, a chimeric lysin with a putative calcium-binding motif, was used as a model for in vitro and in vivo investigations. Methods The concentration of calcium bound to ClyF was determined by atomic absorption spectrometry. The influence of calcium on the structure, activity and host range of ClyF was assessed by circular dichroism and time–kill assays. The bactericidal activity of ClyF was evaluated in various sera and a mouse model of Streptococcus agalactiae bacteraemia. Results ClyF has a highly negatively charged surface around the calcium-binding motif that can bind extra calcium, thereby increasing the avidity of ClyF for the negatively charged bacterial cell wall. In line with this, ClyF exhibited significantly enhanced staphylolytic and streptolytic activity in various sera containing physiological calcium, including human serum, heat-inactivated human serum, mouse serum and rabbit serum. In a mouse model of S. agalactiae bacteraemia, intraperitoneal administration of a single dose of 25 μg/mouse ClyF fully protected the mice from lethal infection. Conclusions The present data collectively showed that physiological calcium improves the bactericidal activity and host range of ClyF, making it a promising candidate for the treatment of infections caused by multiple staphylococci and streptococci.
With the increasing morbidity and mortality rates associated with multidrug-resistant bacteria, interest in bacteriophage therapy has been revived. However, bacterial resistance to phage infection threatens the usefulness of phage therapy, especially its inclusion in modern medicine. Multidrug-resistant Acinetobacter baumannii is a top-priority pathogen requiring urgent intervention and new therapeutic approaches, such as phage therapy. Here, we experimentally adapted A. baumannii WHG40004 to its lytic phage P21 and thereafter isolated a phage-resistant bacterial mutant, named Ev5-WHG. We then aimed to identify potential agents to aid phage killing of Ev5-WHG by analyzing its genome and that of the wild-type strain. The enriched Gene Ontology (GO) analysis based on genetic alterations in minor alleles and mutations showed that pathways such as zinc ion transport and cell membrane synthesis could play certain roles in phage resistance. Remarkably, the combination of zinc acetate and P21 showed increased bactericidal effect on Ev5-WHG. Significantly also, we showed that P21 completely prevented the growth of wild-type WHG40004 in the presence of antibiotics (meropenem and imipenem). The results from this study indicate that the analysis of phage resistance signatures during adaptation of bacteria to a lytic phage can inform the choice of agents to work cooperatively with phage to limit and/or reverse resistance. This approach could be important for guiding future successful phage therapy. IMPORTANCE Bacteriophages have proven very useful as alternative therapeutic agents in combating multidrug-resistant bacterial infections; however, bacterial resistance to phages threatens their use. In this study, we showed a new strategy of leveraging genetic signatures that accompany phage resistance in bacteria to predict agents that can be used with lytic phages to combat multidrug-resistant Acinetobacter baumannii. Significantly, this approach was helpful in suggesting the use of zinc acetate to reduce resistance in phage-resistant bacteria, as well as the use of phage with antibiotics meropenem and imipenem to prevent resistance in a wild-type strain of multidrug-resistant A. baumannii. The approach of this study will be helpful for improving the outcome of phage therapy and in overcoming antimicrobial resistance.
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