key: cord-0868562-sbon3aes authors: Mok, Chee Keng; Ng, Yan Ling; Ahidjo, Bintou Ahmadou; Hua Lee, Regina Ching; Choy Loe, Marcus Wing; Liu, Jing; Tan, Kai Sen; Kaur, Parveen; Chng, Wee Joo; Wong, John Eu-Li; Wang, De Yun; Hao, Erwei; Hou, Xiaotao; Tan, Yong Wah; Mak, Tze Minn; Lin, Cui; Lin, Raymond; Tambyah, Paul; Deng, JiaGang; Hann Chu, Justin Jang title: Calcitriol, the active form of vitamin D, is a promising candidate for COVID-19 prophylaxis date: 2020-06-22 journal: bioRxiv DOI: 10.1101/2020.06.21.162396 sha: a3fad86312bced400081db998fe7cd87ca74178c doc_id: 868562 cord_uid: sbon3aes COVID-19, the disease caused by SARS-CoV-2 (1), was declared a pandemic by the World Health Organization (WHO) in March 2020 (2). While awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4). To expand this arsenal, we screened 4 compound libraries: a United States Food and Drug Administration (FDA) approved drug library, an angiotensin converting enzyme-2 (ACE2) targeted compound library, a flavonoid compound library as well as a natural product library. Of the 121 compounds identified with activity against SARS-CoV-2, 7 were shortlisted for validation. We show for the first time that the active form of Vitamin D, calcitriol, exhibits significant potent activity against SARS-CoV-2. This finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of SARS-CoV-2 patients. Abstract: COVID-19, the disease caused by SARS-CoV-2 (1), was declared a pandemic by the World Health Organization (WHO) in March 2020 (2) . While awaiting a vaccine, several antivirals are being used to manage the disease with limited success (3, 4) . To expand this arsenal, we screened 4 compound libraries: a United States Food and Drug Administration (FDA) approved drug library, an angiotensin converting enzyme-2 (ACE2) targeted compound library, a flavonoid compound library as well as a natural product library. Of the 121 compounds identified with activity against SARS-CoV-2, 7 were shortlisted for validation. We show for the first time that the active form of Vitamin D, calcitriol, exhibits significant potent activity against SARS-CoV-2. This finding paves the way for consideration of host-directed therapies for ring prophylaxis of contacts of SARS-CoV-2 patients. Despite implementation of physical distancing, mask wearing, quarantine and the tireless efforts expended for contact tracing, the rapid transmissibility of SARS-CoV-2 even during the asymptomatic phase has made containment of this virus extremely difficult. The main proposed strategy to curb this pandemic is the implementation of mass vaccination programs. Once a suitable vaccine is discovered, the significant challenges associated with vaccination programs e.g. limitations in manufacturing capabilities and associated costs, are anticipated to significantly affect uptake of vaccinations globally. We therefore propose that ring prophylaxis, which had been previously proposed for influenza pandemics and involves treating close contacts of a confirmed case with an antiviral prophylaxis to further curb community spread (5) , be considered as a viable strategy to reduce transmission of SARS-CoV-2. In an effort to identify potential candidates for SARS-CoV-2 chemoprophylaxis, we performed a virusinduced cytopathic effect (CPE) based screen of several small molecule libraries in SARS-CoV-2infected Vero E6 cells (Fig. 1a) . The African green monkey kidney epithelial Vero E6 cells were used for the screen as these cells are highly susceptible to coronaviruses and exhibit obvious CPE upon infection. A 57-compound natural product library and a library of 462 ACE2 targeted inhibitors (the ACE2 receptor was identified to be necessary for SARS-CoV-2 infection (6)) were used in a preinfection treatment screen to identify potential viral entry inhibitors, while a post-infection treatment screen was performed using both a 500 compound flavonoid library and a 1172 FDA-approved compound libraries in order to identify potential inhibitors targeting post-entry steps of the SARS-CoV-2 replication cycle. For the pre-infection treatment screen, Vero E6 cells were treated with compounds for two hours prior to infection with SARS-CoV-2. The post-infection treatment screen on the other hand was performed by adding compounds to the Vero E6 cells 1 hour post-infection with SARS-CoV-2. Compounds which showed less than 50% CPE compared to the 0.1%DMSO vehicle control with SARS-CoV-2 infection were identified as hits (Tables S1-S3) . Using this method, we identified 31 compounds from the pre-infection treatment screen and 90 compounds from the post-infection treatment screen with activity against SARS-CoV-2 (Table S4) . As expected, our hit list included the tyrosine kinase inhibitors masitinib and imatinib mesylate (7), the antiretroviral drug lopinavir(8) and the calpain inhibitor calpeptin (9) -all compounds reported to inhibit SARS-CoV, SARS-CoV-2 or MERS-CoV. This provides the robustness and confidence of our primary screen for potential antivirals against SARS-CoV-2. Of these primary hits, 7 compounds were selected for downstream validation (Table 1 ). These included 3 compounds from the pre-infection treatment screen (citicoline, pravastatin sodium and tenofovir alafenamide) and four compounds from the post-infection treatment screen (imatinib mesylate, calcitriol, dexlansoprazole, and prochlorperazine dimaleate). These compounds were selected based on level of CPE inhibition in the primary screens (Fig. 1b) , known mechanism of action and existing FDA approval or Generally Recognized as Safe (GRAS) status. FDA approval was considered an important factor as pre-existing data on safety and dosage would allow expedited decisions to be made regarding the potential use of these compounds in vulnerable populations to stymie the current pandemic. Validation assays to determine changes in infectious virus titres upon treatment was carried out by testing selected hit compounds in dose-dependent assays in Vero E6 to confirm the primary screen observation and also in the human hepatocarcinoma HuH7 cell line as the latter cell line expresses high levels of the ACE2 receptor (10) and supports replication of coronaviruses (11) . Cell viability assays were also carried out to ensure that reduction of SARS-CoV-2 titres was not due to cytotoxic effects of the compounds on host cells. CC50, IC50 were obtained for each of the 7 compounds in Vero E6 cells (Table S5 ) and HuH7 cells (Table S6) (Table S6 ). Given that the HuH7 cell line is a hepatocarcinoma cell line and therefore not the first point of entry for SARS-CoV-2 in humans, we decided to test the three most promising compounds (imatinib mesylate, citicoline and calcitriol) against SARS-CoV-2 in the primary human nasal epithelial cell line (hNEC) that is a known in vivo target of SARS-CoV-2 (12) (Fig. 1a) . Despite its significant activity in the continuous cell lines (Vero E6 and HuH7), in hNECs, imatinib mesylate only displayed a 0.2 log 10 reduction in viral titre (Fig. 3) . Interestingly, out of the three compounds only calcitriol proved effective against SARS-CoV-2 with a reduction of 0.69 log 10 in viral titre (Fig. 3 ). While recent data has shown that vitamin D levels are negatively associated with morbidity and mortality of COVID-19 cases (13, 14) , this is the first report of a direct inhibitory effect of calcitriol on SARS-CoV-2. Vitamin D is well known to modulate host immune responses through the production of the antimicrobial peptides such as cathelicidin to promote autophagy (15) . It has proven essential for host defenses against many intracellular pathogens including respiratory pathogens such as Mycobacterium tuberculosis, and has been shown to also possess anti-inflammatory properties (15) . A recent study by Smith and colleagues (16) showed an association between vitamin D deficiency and SARS-CoV-2 infection and COVID-19 associated mortality. The authors speculated that vitamin D supplementation could protect against SARS-CoV-2 infection and improve patient disease outcomes (16) , and our finding certainly provides credence to this hypothesis. Given that calcitriolmediated inhibition occurred upon post-treatment of Vero E6 cells and hNECs, it is likely that its mechanism of antiviral action targets the post-entry phase of viral replication. Use of Host-directed therapies (HDTs) for prevention of infections is certainly not a new idea. Most of these therapies however rely mainly on the use of vaccines, convalescent plasma and monoclonal antibodies (17, 18) . Small molecule HDTs have been used adjunctively for diseases such as tuberculosis (19) and have been proposed for viral pandemics (5) . This strategy would overcome some of the costs and challenges associated with antiviral production, including the emergence of drug resistance (20) . Vitamin D is a dietary supplement that is cheap and widely available even in low and middle income countries and is converted by the liver and kidneys into the active compound calcitriol (21, 22) . It is however important that our findings be confirmed in vivo as well as in clinical trials in order to assess efficacy, optimal dosage, treatment duration, toxicity and safety of calcitriol. Given the high transmissibility of SARS-CoV-2 globally (23), if these findings can be replicated in clinical trials, calcitriol may certainly prove to be an effective tool in the effort to control the pandemic while waiting for an effective vaccine to be rolled out globally. At the very least, these findings certainly pave the way for consideration of host-directed therapies for ring prophylaxis of contacts of SARS-CoV-2 patients. Coronaviridae Study Group of the International Committee on Taxonomy of V. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 WHO. 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