key: cord-0308371-xi0cfpo8
authors: Iaconis, Daniela; Talarico, Carmine; Manelfi, Candida; Cesta, Maria Candida; Zippoli, Mara; Caccuri, Francesca; Matusali, Giulia; Bordi, Licia; Scorzolini, Laura; Bucci, Enrico; Caruso, Arnaldo; Nicastri, Emanuele; Allegretti, Marcello; Beccari, Andrea Rosario
title: Characterization of raloxifene as potential pharmacological agent against SARS-CoV-2 and its variants
date: 2021-10-24
journal: bioRxiv
DOI: 10.1101/2021.10.22.465294
sha: 0c54d052a062291bfc65b38850b756c1d99ec1e8
doc_id: 308371
cord_uid: xi0cfpo8

The new coronavirus that emerged, called SARS-CoV-2, is the causative agent of the COVID-19 pandemic. The identification of potential drug candidates that can rapidly enter clinical trials for the prevention and treatment of COVID-19 is an urgent need, despite the recent introduction of several new vaccines for the prevention and protection of this infectious disease, which in many cases becomes severe. Drug repurposing (DR), a process for studying existing pharmaceutical products for new therapeutic indications, represents one of the most effective potential strategies employed to increase the success rate in the development of new drug therapies. We identified raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a potential pharmacological agent for the treatment of COVID-19 patients. Following a virtual screening campaign on the most relevant viral protein targets, in this work we report the results of the first pharmacological characterization of raloxifene in relevant cellular models of COVID-19 infection. The results obtained on all the most common viral variants originating in Europe, United Kingdom, Brazil, South Africa and India, currently in circulation, are also reported, confirming the efficacy of raloxifene and, consequently, the relevance of the proposed approach. Taken together, all the information gathered supports the clinical development of raloxifene and confirms that the drug can be proposed as a viable new option to fight the pandemic in at least some patient populations. The results obtained so far have paved the way for a first clinical study to test the safety and efficacy of raloxifene, just concluded in patients with mild to moderate COVID-19.

The new coronavirus that emerged, called SARS-CoV-2, is the causative agent of the pandemic. The identification of potential drug candidates that can rapidly enter clinical trials for the 24 prevention and treatment of COVID-19 is an urgent need, despite the recent introduction of several 25 new vaccines for the prevention and protection of this infectious disease, which in many cases 26 becomes severe. Drug repurposing (DR), a process for studying existing pharmaceutical products for 27 new therapeutic indications, represents one of the most effective potential strategies employed to 28 increase the success rate in the development of new drug therapies. We identified raloxifene, a known , SARS-CoV-2 is able to cause severe acute respiratory illness, multi-organ failure and death, 49 sharing common pathogenetic mechanisms with SARS-CoV and MERS-CoV betacoronavirus [5] . 50 Common symptoms of COVID-19 include fever, sore throat, fatigue, cough, shortness of breath and 51 dyspnea that may eventually progress towards acute respiratory distress syndrome, with the 52 involvement of other systems/organs (e.g., heart, liver and kidneys) [6, 7] and up to the death in the 53 most critical cases. About 80% of patients have mild to moderate disease, 14% have severe disease, 54 and 6% become critical (namely, they develop respiratory failure, septic shock, and/or multiple organ 55 dysfunction/failure) (www.ecdc.europa.eufifth update, 2 March 2020). As of September 6 th, 2021, 56 SARS-CoV-2 infection led to more than 4,5 million deaths worldwide (https://covid19.who.int/ ). To 57 date, notwithstanding the advent of vaccine programs and constant social distancing interventions, it 58 is believed that the virus is likely or very likely to become endemic [8, 9] . In addition, the emerging 59 of SARS-CoV-2 variants raises great concern for vaccine efficacy, reinfection events and increased 60 transmissibility and disease severity. As the virus started to spread around the world, a mutated spike 61 SARS-CoV-2 variant (D614G) emerged and was associated with increased infectivity, becoming the 62 predominant variant in Europe and worldwide without any increase in disease severity [10] [11] [12] [13] . In 63 recent months, other variants were defined as "variants of concern" (VOC given to research to increase the availability of broad-spectrum drugs or vaccines for long-term 72 prevention, treatment and control of COVID-19, with the final goal to find new interventions and 73 cures to complement vaccine programs. To identify potential therapeutic targets, one of the main 74 studied mechanisms is the virus entry machinery, and several preclinical and clinical trials are 75 ongoing to find new inhibitors of clinical relevance [22] . The entry machinery involves two key host 76 proteins: the angiotensin-converting enzyme 2 (ACE2) and the cell surface transmembrane protease 77 serine 2 (TMPRSS2) [23, 24] . In addition, also Neuropilin1 (NRP-1) has been recognized as an 78 important receptor whose inhibition reduces SARS-CoV-2 entry and infectivity [25, 26] .

Moreover, recent evidence shows that Nuclear Receptors (NRs), and in particular the sex hormone 80 receptors, like estrogen and androgen receptors, could be involved in the outcome of COVID-19.

These receptors regulate the viral entry protein expression and activity [27] [28] [29] . Additionally, a 82 protective effect of estrogens in the progression of COVID-19 infection has been associated with their 83 role in regulation of innate and adaptive immune responses, as well as in the control of the cytokine 84 storm [30] [31] [32] [33] [34] , whereas activation of androgen receptors seems to correlate with the worse COVID- 85 19 clinical outcome observed in men compared to women [29, [35] [36] [37] .

Recently, several molecules with potential efficacy against SARS-CoV-2 were selected from an Raloxifene is a drug registered in Europe and US for the treatment and prevention of osteoporosis in 97 postmenopausal women, and for the reduction of the risk of invasive breast cancer in postmenopausal 98 women [48, 49] . It is known to act as an agonist in the bone, liver and cardiovascular system and as 99 antagonist in human breast and uterine tissues [50] [51] [52] , and tissue specificity is relevant for its use in Gibco, Thermo-Fisher) at 37°C in a humidified atmosphere of 5% CO2. Cells were seeded at a density of 5x10 5 cells/well in a 12-well plate and incubated at 37°C for 24 h.

Supernatants from infected cells were serially diluted in DMEM without FBS and added to the cells. 

At first, a standard assay was carried out to measure the activity of raloxifene on cell metabolism. To CoV-2 genome copy number already at 10 µM and a 99-fold reduction at 15 µM ( Figure 1E ).

Accordingly, western blot (WB) analysis showed a dose-dependent inhibition of SARS-CoV-2 upon 291 raloxifene treatment with 43% reduction of NP viral protein expression at a concentration of 5 µM, 292 with 65% and 97% reduction at 10 µM and 15 µM, respectively ( Figure 1F ). Raloxifene IC50 value 293 was calculated to be 3.3 µM. SI was then calculated and found to be 5.6. We then tested the raloxifene 294 activity on Calu-3 cells as a model of human pulmonary cell line. The CC50 value was determined, as 295 above, and found to be 24.4 µM ( Figure 2A ). Next, Calu-3 cells were infected as described above.

Supernatants were collected at 48 h p.i., and tested for viral genome copy numbers by qRT-PCR. As 297 shown in Figure 2B , the treatment significantly reduced the virus yield. In particular, raloxifene were performed at least in three independent replicates and pictures shown are representative. Data are presented as the 325 mean + standard error of the mean *, P < 0.05; **, P < 0.01; ****, P < 0.0001. 326

We then performed a systematic study of the antiviral efficacy of raloxifene on the most common 330 variants on Vero E6 cells (Figure 3) . Different viral strains were used: the wild type isolated in 331 January 2020 from Chinese patient (named Wuhan), two different isolates for the D614G spike 332 variants representing the dominant strains circulating in Europe from April to December 2020 (named 333 GV and D614G) and the variants of major concern originated in UK, Brazil, South Africa and India 334 (named VOC B1.1.7, VOC P.1, VOC B1.351, and VOC B1.617.2, respectively). We first determined 335 the time-window in which CPE appeared for each variant. The CPE was evident at 48h for all the 336 tested strains but VOC B1.1.7 and VOC P.1 variant, for which evident CPE appeared later (56h and 337 72h, respectively). In parallel, uninfected cells were cultured in the presence of different doses of 338 raloxifene to evaluate possible cytotoxicity due to the treatment. In cells treated with drug at 15 M, 339 we observed a reduced percentage of viable cells as revealed by crystal violet staining (82.9 +/-8.69% The characterization was completed testing raloxifene also against all the most common circulating 418 SARS-CoV-2 variants of clinical relevance, confirming that it maintains a high and consistent 419 activity, thus reinforcing the interest on its potential clinical use as antiviral agent in COVID-19 420 patients.

Raloxifene cytotoxicity was assessed with two independent assays: the first measuring the activity of 422 the compound on cell replication, the second on cellular metabolism. With both approaches we found 423 that the CC50 was attested at high micromolar range, which is far from the low micromolar range in 424 which the antiviral activity was observed. The selectivity index (SI) of the drug was superimposable 425 in the two experimental models in the range of 2 to 7. In general, the value of SI for a drug with direct 426 antiviral activity is greater than 1; the higher the SI value, the more effective and safer the drug is.

Some authors [85-87] report a limit value of SI = 4 to define a compound as a good compound with 428 direct antiviral activity. The SI value found in the models is therefore indicative of a molecule with a 429 significant antiviral activity and with an activity/toxicity profile consistent with a possible translation 430 to human clinical trials. In addition, raloxifene is a drug that has been used for a long time, and its 431 safety profile is supported by a huge volume of clinical data from long term treatments [88] [89] [90] . The 432 occurrence of thromboembolic events, even though rare, in patients treated with raloxifene has to be 433 regarded with particular caution due to the high risk of thromboembolic manifestations in COVID These findings on one hand are in agreement with previous papers highlighting the protective effect 452 of estrogen signalling in the context of COVID-19 infection, and on the other hand confirm the 453 specific characteristics of raloxifene as an ideal candidate to put to a test the hypothesis in the clinics 454 due to ist peculiar mechanism within the class of SERMs and its potential ability to exert a pleiotropic 455 effect by targeting viral and host targets with a key role in the disease progression and exacerbation. 

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