key: cord-0782607-20lyfx8z authors: Tito, Annalisa; Colantuono, Antonio; Pirone, Luciano; Pedone, Emilia; Intartaglia, Daniela; Giamundo, Giuliana; Conte, Ivan; Vitaglione, Paola; Apone, Fabio title: A pomegranate peel extract as inhibitor of SARS-CoV-2 Spike binding to human ACE2 (in vitro): a promising source of novel antiviral drugs date: 2020-12-01 journal: bioRxiv DOI: 10.1101/2020.12.01.406116 sha: 56a4aa92bf8414712dd54982a6cab611cfeca320 doc_id: 782607 cord_uid: 20lyfx8z Plant extracts are rich in bioactive compounds, such as polyphenols, sesquiterpenes and triterpenes, with potential antiviral activities. As the dramatic outbreak of the pandemic COVID-19, caused by the SARS-CoV-2 virus, thousands of scientists are working tirelessly trying to understand the biology of this new virus and the disease pathophysiology, with the main goal to discover effective preventive treatments and therapeutic agents. Plant-derived secondary metabolites may play key roles in preventing and counteracting the rapid spread of SARS-CoV-2 infections by inhibiting the activity of several viral proteins, in particular those involved in the virus entry into the host cells and its replication. In this study, by using different in vitro approaches, we uncovered the role of a pomegranate peel extract in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein and the human Angiotensin-Converting Enzyme 2 (ACE2) receptor, and in inhibiting the activity of the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this extract in vivo, our results open up new promising opportunities to employ natural extracts for the development of effective and innovative therapies in the fight against SARS-CoV-2. of several viral proteins, in particular those involved in the virus entry into the host cells and its 29 replication. In this study, by using different in vitro approaches, we uncovered the role of a 30 pomegranate peel extract in attenuating the interaction between the SARS-CoV-2 Spike glycoprotein 31 and the human Angiotensin-Converting Enzyme 2 (ACE2) receptor, and in inhibiting the activity of 32 the virus 3CL protease. Although further studies will be determinant to assess the efficacy of this 33 extract in vivo, our results open up new promising opportunities to employ natural extracts for the 34 Introduction PPE compounds inhibited the virus 3CL protease, suggesting a potential use of the extract as natural 112 remedy to enhance protection against SARS-CoV-2. 113 Dried pomegranate peels were provided by Giovomel, an Italian company producing pomegranate 116 juice. The preparation of the Pomegranate Peel Extract (PPE) was performed by adding 700 mL of a 117 solution ethanol/water (70/30, v/v) to 150 g of dried peels, at 4°C, according to Malviya et al., 2014 45 . 118 The mixture was homogenized 3 min at 1500 rpm and 2 min at 3000 rpm by using a 119 Grindomix GM 300 knife mill (Retsch GmbH, Haan, Germany). The resulting suspension was left 120 under stirring at 150 rpm for 2 h at 25°C, avoiding light exposure. The suspension was then 121 centrifuged at 6300 rpm for 10 min at 4°C. The supernatant was filtered through a filter paper 122 Calibration curves were constructed in the linearity ranges of 1-50 µg/mL for PC and 0.1-5 µg/mL 140 for EA, GA. Metabolite identification was performed by using exact mass values up to the fifth 141 decimal digit with mass tolerance ± 5 ppm. Table 1 reports the polyphenols identified in PPE and 142 individual molecular formula, retention time, theoretical mass, experimental mass and error. The 143 amount of each compound in the extract was determined by using PC (1:1000 dilution in PBS + BSA 1%). After 2h, the plate was washed 3 times and incubated with 5 236 µg/mL of peroxidase-conjugated streptavidin for 1 hour at room temperature. After 3 washes, 0.5 237 mg/mL of OPD in 50 mM citrate buffer + 0.012% H2O2 was added to each well and the absorbance 238 was measured at 490nm by the microplate reader Victor Nivo (Perkin Elmer). 239 To measure the activity of the viral 3CL protease in the presence of PPE extract we used the Untagged 241 All the measures were expressed as means ± standard deviations (SD) of three independent 249 experiments. A paired-samples t-test was conducted by Microsoft Excel; a p value lower than 0.05 250 was considered statistically significant. 251 Chemical characterization of PPE 254 The concentration of polyphenols in PPE is reported in Table 2 To assess whether PPE had an inhibitory activity on S/ACE2 binding, we used a SARS-CoV-2 266 inhibitor screening kit by Adipogen. PPE, used at three concentrations ranging from 0.04 mg/mL to 267 1 mg/mL, inhibited the interaction between S and ACE2 up to 74%, and this effect was dose 268 dependent ( Figure 1) . As positive control, we used AC384, a monoclonal antibody that inhibited the 269 binding between S and ACE2 by specifically recognizing ACE2 itself, accordingly to the 270 manufacturer's instruction. 271 To provide insights into which of the PPE polyphenols were relevant for that inhibition, the three 272 most abundant components of PPE, i.e. PC, EA and GA, were individually tested, at the same 273 concentrations as present into 0.04 mg/mL PPE. The results in Table 3 showed that PC most affected the binding between S and ACE2 by exerting 49% inhibition, followed by EA with 36% inhibition, 275 whereas GA did not have any effect. 276 To further investigate on the pomegranate compound binding capacity, the chemical interactions 277 between the extract and S, and between the extract and ACE2, were analysed by MicroScale 278 Thermophoresis (MST) experiments (Figure 2, S1 and S2) . The results showed that the PPE bound 279 both the proteins (Figure 2) , even though the interaction with S was 10 folds stronger than that to 280 ACE2. Moreover, we observed that the binding of PPE compounds to S was mostly due to a high 281 affinity towards the Receptor Binding Domain (RBD) of the protein, as the chemical interaction to 282 this domain was more similar to that calculated for the full-length protein. The biochemical data prompted us to investigate on the capacity of PPE to effectively inhibit the 284 interaction between S and ACE2 in a cellular model. To do that, we used a system based on a Spike-285 carrying Lentivirus, infecting human renal cells (HK2), already known to express ACE2 51 . As control 286 we used a lentivirus that did not carry S, but the Vesicular Stomatitis Virus G (VSVG) protein, thus To investigate whether PPE could regulate host genes involved in the virus uptake, we measured the 296 expression level of ACE2 and TMPRSS2 genes in HK2 cells treated with the extract for 72 h. As 297 reported in Figure 4 , the gene expression analysis showed that the treatment of HK2 cells with the PPE at 0.04 mg/mL reduced the level of ACE2 and TMPRSS2 gene expression by 30% and 70% 299 respectively. This suggested that PPE, besides Spike/ACE2 binding inhibition, was able to 300 downregulate the expression of two genes responsible for the virus access into the cells. 301 As the expression of TMPRSS2 was mainly regulated by androgens 52,53 , we analysed if PPE inhibited 302 the 5α-Reductase activity, primary enzyme involved in DiHydroTestosterone (DHT) synthesis. As 303 shown in Figure 5 , PPE at 0.04mg/mL reduced the activity of the 5α-Reductase by 65% in Human 304 Follicle Dermal Papilla cells (HFDPC), after stimulation by testosterone. This effect was similar to 305 that obtained by finasteride, used as positive control 54 . 306 The regulation of the 3CL protease, one of the main proteins involved in the virus replication, by the Tables 555 556 Table 1 *Expressed as sum of mg of punicalagin equiv. + mg of ellagic acid equiv. + mg of gallic acid 566 equiv.;