Filippini, F. | bioRxiv Science

bioRxiv2024

Snapshots of Pseudomonas aeruginosa SOS response activation complex reveal structural prerequisites for LexA engagement and cleavage

Antimicrobial resistance represents a major threat to human health and Pseudomonas aeruginosa stands out among the pathogens responsible for this emergency. The SOS response to DNA damage plays a pivotal role in bacterial evolution, driving the development of resistance mechanisms and influencing the adaptability of bacterial populations to challenging environments, particularly in the context of antibiotic exposure. Recombinase A (RecA) and the transcriptional repressor LexA are the key players that orchestrate this process, determining either the silencing or the active transcription of the genes under their control. By integrating state-of-the-art structural approaches with binding and functional assays in vitro, we elucidated the molecular events governing the SOS response activation in P. aeruginosa, focusing on the RecA-LexA interaction. Our findings identify the conserved determinants and strength of the interactions that let RecA trigger the autocleavage and inactivation of the LexA repressor. These results provide the groundwork for designing novel antimicrobial strategies and for exploring the potential translation of Escherichia coli-derived approaches, to address the health-threatening implications of bacterial infections. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/585941v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@2fe1e4org.highwire.dtl.DTLVardef@19741e1org.highwire.dtl.DTLVardef@1664ddborg.highwire.dtl.DTLVardef@18195cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity

The pandemic caused by the SARS-CoV-2 has created the need of compounds able to interfere with the biological processes exploited by the virus. Doxycycline, with its pleiotropic effects, including anti-viral activity, has been proposed as a therapeutic candidate for COVID-19 and about twenty clinical trials have started since the beginning of the pandemic. To gain information on the activity of doxycycline against SARS-CoV-2 infection and clarify some of the conflicting clinical data published, we designed in vitro binding tests and infection studies with a pseudotyped virus expressing the spike protein, as well as a clinically isolated SARS-CoV-2 strain. Doxycycline inhibited the transduction of the pseudotyped virus in Vero E6 and HEK-293 T cells stably expressing human receptor angiotensin-converting enzyme 2 but did not affect the entry and replication of SARS-CoV-2. Although this conclusion is apparently disappointing, it is paradigmatic of an experimental approach aimed at developing an integrated multidisciplinary platform. To avoid wasting precious time and resources we believe very stringent experimental criteria are needed in the preclinical phase, including infectious studies with SARS-CoV-2 in the platform before moving on to [failed] clinical trials. Author SummaryThe pandemic caused by the SARS-CoV-2 virus has created a completely unusual situation in rapidly searching for compounds able to interfere with the biological processes exploited by the virus. This new scenario has substantially changed the timing of drug development which has also resulted in the generation of controversial results, proving that the transition from computational screening to the clinical application requires great caution and careful studies. It is therefore necessary to establish new paradigms for evaluating the efficacy of a potential active molecule. We set up a preclinical platform aimed at identifying molecules active against SARS-CoV-2 infection developing a multidisciplinary approach based on very stringent experimental criteria, comprising in-silico studies, in vitro binding tests and infection studies with pseudovirus expressing the spike protein as well as clinically isolated SARS-CoV-2 strains. We focused our attention on doxycycline which has been suggested as potential therapeutic candidate for treating COVID-19 and is currently employed in about twenty clinical trials. Doxycycline resulted effective in inhibiting the transduction of pseudovirus but it did not affect the entry and replication of SARS-CoV-2. The results obtained underline the need to define more stringent and controlled pharmacological approaches before wasting precious time and resources with clinical trials.

pharmacology and toxicology↗