Biology subjects

Curran, J. F.

Publications and source records attributed to Curran, J. F..

4 recordsLinked to original sources

bioRxiv2021

Analyses of the 5-prime Ends of Escherichia coli ORFs

Sequence biases at 5 ends of coding sequences differ from those of the remainder of ORFs, reflecting differences in function. Internal sequence biases promote translational efficiency by several mechanisms including correlating codon usage and tRNA concentration. However, the early region may also facilitate translational initiation, establishment of the reading frame, and polypeptide processing. Here we examine the beginnings of the ORFs of an Escherichia coli K12 reference genome. The results extend previous observations of A-richness to include an overabundance of the AAA triplet in all reading frames, consistent with the hypothesis that the beginnings of ORFs contribute to initiation site accessibility. Results are also consistent with the idea that the first two amino acids are under selection because they facilitate solvation of the amino-terminus at the end of the ribosomal exit channel. Moreover, serine is highly overrepresented as the second amino acid, possibly because it can facilitate removal of the terminal formylmethionine. Non-AUG initiation codons are known to be less efficient than AUG at directing initiation, presumably because of relatively weak base pairing to the initiator-tRNA. But non-UAG initiation codons are not followed by unusual 3 nearest neighbor codons. Moreover, the four NUG initiation codons do not differ in their propensity to frameshift in an assay known to be sensitive to base pair strength. Altogether, these data suggest that the 5 ends of ORFs are under selection for several functions, and that initiation codon identity may not be critical beyond its role in initiation.

molecular biology↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of nsp5 Main Protease

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5,000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

biochemistry↗

bioRxiv2021

Identification of SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of the nsp14 RNA Cap Methyltransferase

The COVID-19 pandemic has presented itself as one of the most critical public health challenges of the century, with SARS-CoV-2 being the third member of the Coronaviridae family to cause fatal disease in humans. There is currently only one antiviral compound, remdesivir, that can be used for the treatment of COVID-19. In order to identify additional potential therapeutics, we investigated the enzymatic proteins encoded in the SARS-CoV-2 genome. In this study, we focussed on the viral RNA cap methyltransferases, which play a key role in enabling viral protein translation and facilitating viral escape from the immune system. We expressed and purified both the guanine-N7 methyltransferase nsp14, and the nsp16 2-O-methyltransferase with its activating cofactor, nsp10. We performed an in vitro high-throughput screen for inhibitors of nsp14 using a custom compound library of over 5,000 pharmaceutical compounds that have previously been characterised in either clinical or basic research. We identified 4 compounds as potential inhibitors of nsp14, all of which also show antiviral capacity in a cell based model of SARS-CoV-2 infection. Three of the 4 compounds also exhibited synergistic effects on viral replication with remdesivir.

bioengineering↗

bioRxiv2021

Identifying SARS-CoV-2 Antiviral Compounds by Screening for Small Molecule Inhibitors of Nsp14/nsp10 Exoribonuclease

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

biochemistry↗