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ACS central science ; 8(5):527-545, 2022.
Article in English | EuropePMC | ID: covidwho-1871009


Heparan sulfate (HS) is a cell surface polysaccharide recently identified as a coreceptor with the ACE2 protein for the S1 spike protein on SARS-CoV-2 virus, providing a tractable new therapeutic target. Clinically used heparins demonstrate an inhibitory activity but have an anticoagulant activity and are supply-limited, necessitating alternative solutions. Here, we show that synthetic HS mimetic pixatimod (PG545), a cancer drug candidate, binds and destabilizes the SARS-CoV-2 spike protein receptor binding domain and directly inhibits its binding to ACE2, consistent with molecular modeling identification of multiple molecular contacts and overlapping pixatimod and ACE2 binding sites. Assays with multiple clinical isolates of SARS-CoV-2 virus show that pixatimod potently inhibits the infection of monkey Vero E6 cells and physiologically relevant human bronchial epithelial cells at safe therapeutic concentrations. Pixatimod also retained broad potency against variants of concern (VOC) including B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), and B.1.1.529 (Omicron). Furthermore, in a K18-hACE2 mouse model, pixatimod significantly reduced SARS-CoV-2 viral titers in the upper respiratory tract and virus-induced weight loss. This demonstration of potent anti-SARS-CoV-2 activity tolerant to emerging mutations establishes proof-of-concept for targeting the HS–Spike protein–ACE2 axis with synthetic HS mimetics and provides a strong rationale for clinical investigation of pixatimod as a potential multimodal therapeutic for COVID-19. Heparan sulfate (HS) has emerged as a SARS-CoV-2 coreceptor. Pixatimod (PG545), an HS mimetic, inhibits infectivity of multiple variants offering a novel therapeutic approach against COVID-19.

Nat Commun ; 12(1): 3431, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1262001


The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.

Reverse Genetics , SARS-CoV-2/genetics , Amino Acid Sequence , Animals , Base Sequence , Chlorocebus aethiops , Culicidae/virology , Furin/metabolism , Genome, Viral , HEK293 Cells , Humans , Mice , Mutation/genetics , NIH 3T3 Cells , Polymerase Chain Reaction , RAW 264.7 Cells , Receptors, Virus/metabolism , Vero Cells , Viral Proteins/chemistry , Virus Replication
Front Microbiol ; 12: 625136, 2021.
Article in English | MEDLINE | ID: covidwho-1110305


Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 and is capable of human-to-human transmission and rapid global spread. The rapid emergence and global spread of SARS-CoV-2 has encouraged the establishment of a rapid, sensitive, and reliable viral detection and quantification methodology. Here, we present an alternative assay, termed immuno-plaque assay (iPA), which utilizes a combination of plaque assay and immunofluorescence techniques. We have extensively optimized the conditions for SARS-CoV-2 infection and demonstrated the great flexibility of iPA detection using several antibodies and dual-probing with two distinct epitope-specific antibodies. In addition, we showed that iPA could be utilized for ultra-high-throughput viral titration and neutralization assay within 24 h and is amenable to a 384-well format. These advantages will significantly accelerate SARS-CoV-2 research outcomes during this pandemic period.

Environ Res ; 196: 110944, 2021 05.
Article in English | MEDLINE | ID: covidwho-1108272


The COVID-19 pandemic has revealed gaps in our understanding of safe, effective and efficient means of disinfecting high use public spaces. Whilst this creates an opportunity for development and application of innovative approaches such as unmanned aerial vehicle (UAV) based disinfection, unregulated outdoor disinfection using chlorine has led to environmental and public health risks. This study has quantified the efficiency, safety and efficacy of UAV-based spraying of aqueous ozone. Optimised UAV flight characteristics of 4.7 km/h at 1.7 m elevation spraying 2.4 L/min were able to provide >97% and >92% coverage of a 1 m and 2 m wide swath respectively. During spraying operations using 1 mg/L aqueous ozone, atmospheric concentrations of ozone remained within background levels (<0.04 ppm). Highly efficient inactivation of two different isolates of SARS-CoV-2 virus was achieved at aqueous ozone concentrations of 0.75 mg/L after an incubation period of only 5 min, with 0.375 mg/L achieving 82-91.5% inactivation in this time. Exposure of diamondback moth larvae and parasitic wasps to 1 mg/L aqueous ozone did not significantly affect their survivorship. These results indicate for the first time that aqueous ozone may provide the required balance between human and environmental safety and viral inactivation efficacy for targeted application in high risk outdoor settings.

COVID-19 , Disinfectants , Ozone , Disinfection , Humans , Pandemics , SARS-CoV-2