Doxycycline Inhibition of a Pseudotyped Virus Transduction Does Not Translate to Inhibition of SARS-CoV-2 Infectivity

Abstract

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.

Keywords: COVID-19; SARS-CoV-2; doxycycline; in vitro; spike protein; surface plasmon resonance; tetracyclines.

Figure 1

Doxycycline inhibited the transduction of pseudotyped retroviral vector exposing the SARS-CoV-2 S protein. (A) Representative image of two isolated pseudotyped retrovirus particles exposing the SARS-CoV-2 S protein. CA, capsid; EN, envelope; MA, matrix; spikes are indicated by red arrowheads. Scale bar, 50 nm. Dose-response effect of doxycycline in VeroE6 (B) and HEK293-ACE2 (C) cells. The y-axis shows the mean ± SD percentage of GFP-transduced cells in relation to control cells. The top limit was set as the average percentage for the vehicle-only control of this assay. Effects of 1 or 100 µM doxycycline (D) and 1 or 100 µM gentamicin (E) on transduction of the pseudotyped retroviral vector with SARS-CoV-2 S protein in Vero E6 and HEK293-ACE cells. Data are means ± SD of the percentage of GFP-transduced cells in relation to control cells transduced with vehicle only (dotted line). The percentage of Vero E6 cells transduced with the retroviral vector without SARS-CoV-2 S protein (No-Spike) is reported as negative control. **** p < 0.0001 vs. vehicle according to one-way ANOVA and Bonferroni’s post hoc test. (F) Representative fluorescence microscopy images of HEK293-ACE2 cells infected with the retroviral vector and treated or not with gentamicin or doxycycline. Scale bar, 100 µm.

Figure 2

Surface plasmon resonance analysis showing no inhibitory effect of doxycycline on the ACE2-RBD interaction. ACE2-RBD interaction was evaluated either by flowing ACE2 (10 nM) over immobilized RBD (A) or, vice versa, RBD (60 nM) over immobilized ACE2 (B). This interaction was evaluated either in the absence or presence of 100 μM doxycycline. In particular, we preincubated the proteins for 60 min at room temperature with doxycycline or its vehicle, and then injected the mixture over the chip-immobilized protein binding partners for 300 s, as indicated. The graphs show the sensorgrams after subtraction of the SPR signal on reference surfaces (anti-Fc antibody for RBD; or anti-Flag antibody for ACE2). These are representative sensorgrams from one experimental session. Results were similar in three independent sessions.

Figure 3

Doxycycline did not inhibit SARS-CoV-2 authentic virus replication. Vero E6 cells were pretreated for 4 h with doxycycline or gentamicin (100 μM), then infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 1 h at 37 °C with doxycycline or gentamicin (100 μM). Cells were then washed and cultured for 48 h in fresh medium containing doxycycline or gentamicin (100 μM). Non-infected cells (NI) or cells infected without doxycycline or gentamicin treatment (SARS-CoV-2) were used as controls. (A) Cells were imaged with an optical microscope to detect typical SARS-CoV-2-induced cytolytic effects (original magnification 10×). (B) Viral yield was quantified in the cell supernatant by qRT-PCR. (C) Quantification of SARS-CoV-2 genomes at the intracellular level by qRT-PCR. Data are means ± SD of at least three independent replicates.