Biology subjects

Ashhurst, A.

Publications and source records attributed to Ashhurst, A..

4 recordsLinked to original sources

bioRxiv2021

Discovery of Antiviral Cyclic Peptides Targeting the Main Protease of SARS-CoV-2 via mRNA Display

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

biochemistry↗

bioRxiv2020

Potent in vitro anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC50 values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.

biochemistry↗

bioRxiv2020

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein using mRNA Display

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (KD) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect spike protein in solution by ELISA, and the co-crystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a {beta}-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD and the ligands discovered in this work may find future use as reagents for diagnostic applications.

biochemistry↗

bioRxiv2020

Mucosal vaccination with CysVac2/Advax induces Th17 lung-resident CD4+ T cells and interleukin-17-dependant protection against Mycobacterium tuberculosis infection

The development of effective vaccines against bacterial lung infections requires the induction of protective, pathogen-specific immune responses without deleterious inflammation within the pulmonary environment. Here, we made use of a polysaccharide-adjuvanted vaccine approach to elicit resident pulmonary T cells to protect against aerosol Mycobacterium tuberculosis infection. Intratracheal administration of the multistage fusion protein CysVac2 and the delta-inulin adjuvant Advax (formulated with a TLR9 agonist) provided superior protection against aerosol M. tuberculosis infection in mice, compared to parenteral delivery. Surprisingly, removal of the TLR9 agonist did not impact vaccine protection despite a reduction in cytokine-secreting T cell subsets, particularly CD4+ IFN-{gamma}+IL-2+TNF+ multifunctional T cells. CysVac2/Advax-mediated protection was associated with the induction of lung-resident, antigen-specific memory CD4+ T cells that expressed IL-17 and ROR{gamma}t, the master transcriptional regulator of Th17 differentiation. IL-17 was identified as a key mediator of vaccine efficacy, with blocking of IL-17 during M. tuberculosis challenge reducing phagocyte influx, suppressing priming of pathogen-specific CD4+ T cells in local lymph nodes and ablating vaccine-induced protection. These findings suggest that tuberculosis vaccines such as CysVac2/Advax that are capable of eliciting Th17 lung-resident memory T cells are promising candidates for progression to human trials. ImportanceMycobacterium tuberculosis, the causative agent of tuberculosis (TB), kills more individuals each year than any other single pathogen. The only approved vaccine, BCG, administered intradermally, is unreliable in protecting against pulmonary TB, therefore a more effective vaccine is critical for global control of the disease. Vaccination in the lung would be a rational way of inducing a local memory immune response to TB, however vaccine platforms would need to deliver antigens to delicate mucosal surfaces without inducing deleterious inflammatory responses. We developed a safe mucosal vaccine which induced protection against TB lung infection in mice by inducing high levels of lung-resident T cells expressing the cytokine IL-17. Removal of IL-17 limited the influx of phagocytic cells to the lung and completely ablated protection afforded by the vaccine. This study provides new insights into mechanisms of protection against M. tuberculosis and provides a promising candidate to protect against TB in humans.

immunology↗