We developed and validated a technology platform for designing and testing peptides inhibiting the infectivity of SARS-CoV-2 spike protein-based pseudoviruses. This platform integrates target evaluation, in silico inhibitor design, peptide synthesis, and efficacy screening. We generated a cyclic peptide library derived from the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor. The cell-free validation process by ELISA competition assays and Surface Plasmon Resonance (SPR) studies revealed that the cyclic peptide c9_05, but not its linear form, binds well to ACE2. Moreover, it effectively inhibited the transduction in HEK293, stably expressing the human ACE2 receptor of pseudovirus particles displaying the SARS-CoV-2 spike in the Wuhan or UK variants. However, the inhibitory efficacy of c9_05 was negligible against the Omicron variant, and it failed to impede the entry of pseudoviruses carrying the B.1.351 (South African) spike. These variants contain three or more mutations known to increase affinity to ACE2. This suggests further refinement is needed for potential SARS-CoV-2 inhibition. Our study hints at a promising approach to develop inhibitors targeting viral infectivity receptors, including SARS-CoV-2’s. This platform also promises swift identification and evaluation of inhibitors for other emergent viruses.

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created an unusual situation, with rapid searches for compounds 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 which can shed light on the mechanisms of action of potential anti-COVID-19 compounds. To avoid wasting precious time and resources, we believe very stringent experimental criteria are needed in the preclinical phase, including infectivity studies with clinically isolated SARS-CoV-2, before moving on to (futile) clinical trials.

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 Summary: The 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.

Testing medical countermeasures for SARS-CoV-2 transmission using vertebrates can be slowed by legislation regulating animal experimentation, high costs, and ethical concerns. To overcome these challenges, we propose a new Caenorhabditis elegans strain that constitutively expresses the human angiotensin-converting enzyme 2 receptor (ACE2). This resulted in significant impairment of reproduction and a defect in pharyngeal function compared to wild-type (WT) worms. SARS-CoV-2 infection was simulated by treating worms with the receptor-binding domain (RBD) of the virus, which caused dose- and time-dependent pharyngeal impairment in ACE2 worms but not in WT worms. The toxicity of RBD was prevented by administering an anti-human ACE2 antibody, demonstrating that interaction with the ACE2 receptor is essential. The ACE2-expressing worm strain was further used for pharmacological research with Raloxifene. In vitro, Raloxifene at 1-3 μM reduced the entry of lentiviral particles carrying the Wuhan variant, B.1.1.7 UK, and B.1.1.529 Omicron strains, into HEK293-ACE2, as well as particles expressing N501Y- or P681H-mutated spike proteins. Raloxifene (0.1-1 μM) completely counteracted RBD toxicity in ACE2 worms, indicating that this strain offers a cost-effective in vivo screening platform for molecules whose effects involve interaction with the ACE2 receptor.

Testing medical countermeasures for SARS-CoV-2 transmission using vertebrates can be hindered by legislation regulating animal experimentation, high costs, and ethical concerns. To overcome these challenges, we propose a new Caenorhabditis elegans strain that constitutively expresses the human angiotensin-converting enzyme 2 receptor (ACE2). This resulted in significant impairment of reproduction and a defect in pharyngeal function compared to wild-type (WT) worms. SARS-CoV-2 infection was simulated by treating worms with the receptor-binding domain (RBD) of the spike protein, which caused dose-dependent and time-dependent pharyngeal impairment in ACE2 worms but not in WT worms. The toxicity of RBD was prevented by administering an anti-human ACE2 antibody, demonstrating that interactions with the ACE2 receptor are essential. The ACE2-expressing worm strain was further used for pharmacological research with Raloxifene. In vitro, 1–3 μM of Raloxifene reduced the entry of lentiviral particles carrying the Wuhan variant and B.1.1.7 UK and B.1.1.529 Omicron strains into HEK293-ACE2, in addition to particles expressing N501Y-mutated or P681H-mutated spike proteins. Raloxifene (0.1–1 μM) completely counteracted RBD toxicity in ACE2 worms, indicating that this strain offers a cost-effective in vivo screening platform for molecules with effects involving interactions with the ACE2 receptor.