key: cord-0276689-ye5f6uw9
authors: Diomede, Luisa; Baroni, Sara; De Luigi, Ada; Piotti, Arianna; Lucchetti, Jacopo; Fracasso, Claudia; Russo, Luca; Bonaldo, Valerio; Panini, Nicolò; Filippini, Federica; Fiordaliso, Fabio; Corbelli, Alessandro; Beeg, Marten; Pizzato, Massimo; Caccuri, Francesca; Gobbi, Marco; Biasini, Emiliano; Caruso, Arnaldo; Salmona, Mario
title: Doxycycline inhibition of a pseudotyped virus transduction does not translate to inhibition of SARS-CoV-2 infectivity
date: 2021-07-30
journal: bioRxiv
DOI: 10.1101/2021.07.30.454436
sha: 31cd26899f5cae5903462b14ee1e37dbdebe0067
doc_id: 276689
cord_uid: ye5f6uw9

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.

The difference in GFP transduction efficiency between the two cell lines cannot be ascribed to a toxic 146 effect of doxycycline, which did not induce significant cytotoxicity in Vero E6 and HEK293-ACE2 147 cells (Fig 2A and Fig 2B) nor affected the proliferation of HEK293-ACE2 cells (Fi 2C). Since gene 148 transfer by retroviral vectors can occur only in cells that are actively replicating at the time of 149 infection, we also investigated whether doxycycline affected the cell cycle of HEK293-ACE2. There 150 was no change in the DNA content in the different phases of cell cycle in cells treated with 1 or 100 151 µM doxycycline at all time points (Fig 2D) . In addition, doxycycline did not affect the level of ACE2 152 expression in HEK293-ACE2 cells (S3 Fig) . 153 These results indicate that doxycycline may reduce cellular entry for a pseudotyped retroviral vector 154 exposing the SARS-CoV-2 S protein and that efficacy may be related to the cell type. 155 156 SPR studies 157 SPR studies were done to determine whether doxycycline reduces retroviral transduction by binding 158 to the S protein and/or ACE2. No evidence of a doxycycline binding, up to 100 μM, to ACE2, S, S1 159 and RBD was obtained in SPR studies using a direct approach (i.e. flowing the drug over immobilized 160 proteins) (Fig 3) . However, the possibility of false negative data cannot be excluded, as SPR has 161 lower sensitivity of SPR when testing small molecules. For this reason, we also employed a different 162 SPR approach to see whether doxycycline inhibited the RBD-ACE2 interaction. This can be detected 163 well by SPR, either flowing ACE2 (10 nM) over immobilized RBD (Fig.4A , purple line, estimated 164 Kd= 0.9 nM) or, viceversa, flowing RBD (60 nM) over immobilized ACE2 (Fig 4B, (Fig 4A and Fig 4B, red lines) , did not affect the binding of the protein 167 with the partner immobilized on the sensor chip (RBD or ACE2, respectively), suggesting that the 168 drug did not occupy the relevant binding sites at a significant extent. 169 170 Effect of doxycycline on authentic SARS-CoV-2 strain replication 9 To determine the ability of doxycycline to counteract the infectivity of SARS-CoV-2, Vero E6 cells 172 were pretreated for 4 h with 100 µM doxycycline or gentamicin before the infection with the authentic 173 SARS-CoV-2 strain at a MOI of 0.01. Cells were then washed and cultured for 48 h in fresh medium 174 containing 100 µM doxycycline or gentamicin. As shown in Fig 5A, SARS-CoV-2 induced cytolytic 175 effects on Vero E6 cells which was not modified by the treatment with doxycycline or gentamicin. 176

Quantification of viral RNA copy number in the cell culture supernatants (Fig 5B) and at intracellular 177 levels (Fig 5C) indicated that doxycycline did not exert any inhibitory effects on viral particles' 178 production and genome expression, respectively. These findings indicated that doxycycline, although 179 effective in the pseudotyped virus transduction assay, did not inhibit SARS-CoV-2 replication. 180 10 

The rapid spread of the pandemic caused by the SARS-CoV-2 virus has created a completely unusual 182 situation in defining the strategies to develop vaccines or antiviral drugs in a broad sense. The 183 pandemic surprised everyone by the speed of its spread and, above all, by the absence of integrated 184 national and international defense strategies (3). 185

The development of medicines usually takes a very long time between conception and the availability 186 to the patient. Still, in COVID-19 case, the time factor was decisive. Therefore, the scientists aimed 187 at developing vaccines and antiviral medicines, reducing the time for their availability as much as 188 possible. Of course, this new scenario has substantially changed the timing of drug development 189 which has also resulted in the generation of many false-negative or false-positive results (3). 190

The possibility of using artificial intelligence to identify potential molecules active against the spread 191 of the pandemic has prompted many groups to carry out in-silico studies and screen entire libraries 192 (24, 25) . 193

In the case of anti-COVID-19 drugs, numerous molecules have been identified through in-silico 194 studies as potentially active, but in reality, the outcome of this kind of approach has not been as 195 successful as expected. Many of the molecules identified in-silico have reported controversial results 196 proving that the transition from in-silico screening to the clinical application requires great caution 197 and careful studies to verify the in vitro efficacy. It is, therefore, necessary to establish new paradigms 198 for evaluating the efficacy of a potential active molecule. 199

As an example, in this paper, we report the controversial results obtained with doxycycline, which in 200 some way echoes those already published in the literature. We demonstrated for the first time that 201 doxycycline significantly inhibited the transduction of a pseudotyped virus on two different cell lines. 202

However, this effect did not translate into the drug's ability to counteract in vitro in Vero E6 cells the 203 entry and replication of authentic SARS-CoV-2 virus. This finding was in contrast with that 204 previously reported by Gendrot and collaborators (16) which, employing Vero E6 cells too, found 205 doxycycline effective in counteracting SARS-CoV-2 infectivity. It cannot be excluded that this 206 11 discrepancy could be due to the different SARS-CoV-2 strains employed to infect the cells. We used 207 a clinically isolated SARS-CoV-2 representative of the most widespread strain in Europe during the 208 first wave of the pandemic which gnomic data are available at EBI under study accession n. 209 PRJEB38101 (26). Gendrot and collaborators employed the IHUMI-3 strain for which genomic data 210 are not available thus making difficult to establish the degree of widespread of the virus and its 211 comparison with other strains. Doxycycline did not interact with relevant binding sites of S or ACE2 212 proteins, as instead suggested by an in-silico study (14). It cannot be excluded that it may affect, at 213 least on the pseudotyped retroviral vector, the integrity of the virus lipidic envelope, suggested to be 214 important for the virus integrity (27). 215

Although the conclusion of our study is somewhat disappointing, it is paradigmatic of an experimental 216 approach aimed at developing an integrated multidisciplinary platform. 217

To avoid wasting precious time and resources we therefore believe that it is necessary to set very 218 stringent experimental criteria in the preclinical phase, including in the platform infectious studies 219 with SARS-CoV-2, before moving on to failed clinical trials. 220

This strategy may help develop a scientifically sound procedure for selecting potentially active 221 molecules at the preclinical stage. 

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of doxycycline to inhibit the ACE2-RBD interaction, we preincubated 10 nM ACE2 (or 60 nM RBD) 353for 60 min at room temperature with or without the drug, and then injected the mixture over chip-354 immobilized RBD (or ACE2). All SPR assays were run at a rate of 30 μL/min at 25°C. The 355 sensorgrams (time course of the SPR signal in RU) were normalized to a baseline of 0.