Biology subjects

Ileperuma, S. M.

Publications and source records attributed to Ileperuma, S. M..

5 recordsLinked to original sources

bioRxiv2026

Discovery of inhibitors of the Pseudomonas aeruginosa NADH:ubiquinone oxidoreductase (NQR) that hinder virulence factors

The growing threat of antimicrobial resistance has created an urgent need to identify novel therapeutic targets in bacteria. The NADH:ubiquinone oxidoreductase (NQR) is a potential target in a number of bacteria that transfers electrons from NADH to ubiquinone while pumping ions from the cytoplasm to the periplasm. In most species, this complex pumps sodium ions, whereas in Pseudomonas aeruginosa it pumps protons, thereby functioning as a member of the electron transport chain. Using a strain of PAO1 with the NQR knocked out, we demonstrate that the NQR complex plays a crucial role in the motility and biofilm development virulence factors in P. aeruginosa. We develop and execute a high-throughput inhibitor screen to identify and confirm compounds that inhibit NADH oxidation by this complex. Using single-particle cryogenic electron microscopy (cryoEM), we determine high-resolution structures of the NQR complex, both inhibitor-free and bound to one of the confirmed hits from our screen, demonstrating that it binds to the ubiquinone binding site. These structures provide insight into conformational dynamics controlled by binding at the ubiquinone site, with potential implications for the coupling between electron transfer and proton pumping in this complex. Biofilm development and motility assays with selected compounds from the screen show that they affect these virulence factors similarly to the NQR knockout.

biochemistry↗

bioRxiv2025

Potent broad-spectrum anti-coronaviral frameshift inhibitors from virtual screen of RNA binding

Coronavirus genomes contain an RNA pseudoknot that directs -1 programmed ribosomal frame-shifting (-1 PRF) to control expression of viral proteins crucial for replication. Ligands that inhibit -1 PRF can thus attenuate viral propagation and have potential as drugs for limiting corona-virus infections. To search for novel small-molecule frameshift inhibitors with anticoronaviral activity, we computationally screened over 14 million compounds for binding to the SARS-CoV-2 pseudoknot, followed by experimental validation of the top hits for inhibition of -1 PRF and viral replication. We identified multiple potent -1 PRF inhibitors, effective at nM concentrations, some of which significantly suppressed SARS-CoV-2 replication in cell culture. Several compounds also inhibited -1 PRF in multiple representative bat coronaviruses, indicating broad-spectrum activity. These results showcase the promise of viral RNA structures like frameshift-stimulatory pseudoknots as targets for broad-spectrum antiviral drugs.

biophysics↗

bioRxiv2021

Small-molecule ligands can inhibit -1 programmed ribosomal frameshifting in a broad spectrum of coronaviruses

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases since 2000 have high-lighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored a group of 6 small-molecule ligands, evaluating their activity against the frameshift signals from a panel of representative bat CoVs--the most likely source of future zoonoses--as well as SARS-CoV-2 and MERS-CoV. We found that whereas some ligands had notable activity against only a few of the frameshift signals, the serine protease inhibitor nafamostat suppressed -1 PRF significantly in several of them, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing the frameshift signal as a viable target for developing pan-coronaviral therapeutics.

biochemistry↗

bioRxiv2020

Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers

The RNA pseudoknot that stimulates -1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frame-shifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5' end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5' end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

biophysics↗

bioRxiv2020

Anti-frameshifting ligand active against SARS coronavirus-2 is resistant to natural mutations of the frameshift-stimulatory pseudoknot

The coronavirus SARS-CoV-2 causing the COVID-19 pandemic uses −1 programmed ribosomal frameshifting (−1 PRF) to control the expression levels of key viral proteins. Because modulating −1 PRF can attenuate viral propagation, ligands binding to the viral RNA pseudoknot that stimulates −1 PRF may prove useful as therapeutics. Mutations in the pseudoknot have been observed over the course of the pandemic, but how they affect −1 PRF and the activity of inhibitors is unknown. Cataloguing natural mutations in all parts of the SARS-CoV-2 pseudoknot, we studied a panel of 6 mutations in key structural regions. Most mutations left the −1 PRF efficiency unchanged, even when base-pairing was disrupted, but one led to a remarkable three-fold decrease, suggesting that SARS-CoV-2 propagation may be less sensitive to modulation of −1 PRF efficiency than some other viruses. Examining the effects of one of the few small-molecule ligands known to suppress −1 PRF significantly in SARS-CoV, we found that it did so by similar amounts in all SARS-CoV-2 mutants tested, regardless of the basal −1 PRF efficiency, indicating that the activity of anti-frameshifting ligands can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of −1 PRF.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗