Targeting ATP2B1 impairs PI3K/Akt/Fox-O3 signaling and reduces SARS-COV-2 replication in vivo

ATP2B1 is a known regulator of calcium (Ca2+) cellular export and homeostasis. Diminished levels of extra- or intra-cellular Ca2+ content have been suggested to block SARS-CoV-2 replication. Here, we demonstrate that a newly nontoxic caloxin-derivative compound (PI-7) inhibits ATP2B1, reduces the extra- and intra-cellular Ca2+ levels and impairs SARS-CoV-2 replication and propagation (VOCs: Delta and Omicron 2), as also measured by inhibition of syncytia in vitro . Furthermore, a FOXO3 transcriptional site of regulation of expression at the 5’ end of the ATP2B1 locus, together with a rare homozygous intronic variant in the ATP2B1 locus (rs11337717; chr12:89643729, T>C), are shown to be associated with severity of COVID19 (symptomatic versus asymptomatic patients). Here, we identify the mechanism of action during SARS-CoV-2 infection, which involves the PI3K/Akt signaling pathway, inactivation of FOXO3 (i.e., phosphorylation), and inhibition of transcriptional control of both membrane and reticulum Ca2+ pumps (ATP2B1 and ATP2A1 [i.e., SERCA1], respectively). The pharmacological action of compound PI-7 on sustaining both ATP2B1 and ATP2A1 expression reduces the intracellular cytoplasmic Ca2+ pool and thus negatively influences SARS-CoV-2 replication and propagation. As compound PI-7 shows a lack of toxicity, its prophylactic use as a therapy against the COVID19 pandemic is here proposed. In brief De Antonellis et al. shows the importance of the Ca2+ channel pump ATP2B1 in the regulation of extracellular and intracellular Ca2+ levels that positively influence SARS-CoV-2 replication in human cells. Our study identifies the mechanism of action of SARS-CoV-2 in the regulation of the expression of ATP2B1 and ATP2A1 loci during infection via FOXO3 transcriptional factor. Furthermore, a small caloxin-derivative molecule (compound PI-7) can inhibit ATP2B1 activity, thus resulting in SARS-CoV-2 impairment. In further support, we have identified a genetic variant within the noncoding upstream region of ATP2B1 in symtomatic patients affected by severe COVID19, thus indicating this polymorphism as a genetic predisposition factor to SARS-CoV-2 infection. Highlights 1. An anti-viral model of network of action for ATP2B1 against SARS-CoV-2 at the intracellular level that involves the PI3K/Akt signaling pathway, inactivation (i.e., phosphorylation) of FOXO3 and its transcriptional control, and inhibition of both membrane and reticulum Ca2+ pumps (i.e., ATP2B1, ATP2A1, respectively). 2. A new drug and its lack of toxicity “compound PI-7”, thus envisioning both preventive and therapeutic applications in patients with COVID-19. 3. The specificity of action in the context of Ca2+ homeostasis is one of the strategies that coronaviruses (including SARS-CoV-2 and any new VOC, including Omicron 2) use to infect host cells and promote organ dysfunction. 4. Therapeutic applications for compound PI-7 against all other viruses belonging to the Coronoviridae family (e.g., SARS-CoV, MERS-CoV), and against the main families of positive sense ssRNA viruses from other hosts (e.g., Nidovirales), as these are all Ca2+ dependent. 5. Identification of a rare homozygous intronic variant in the ATP2B1 locus (rs11337717; chr12:89643729, T>C) that is associated with severity of COVID19 (i.e., symptomatic versus asymptomatic patients). This variant can be used as a marker to identify those patients that might show severe COVID19 following their SARS-COV-2 infection. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was supported by the project CEINGE TaskForce COVID19, code D64I200003800 by Regione Campania for the fight against COVID19 (DGR no. 140; 17 March 2020). We further thank for support the Italian Association for Cancer Research (AIRC) Grant IG no. 22129 (M.Z.), the European School of Molecular Medicine for a doctorate program Fellowship (F.A.) and the Molecular Medicine Doctorate program Fellowship (F.B.), Fondazione Celeghin Italiana (M.Z.), and Ministero dell Universita e della Ricerca Italiana (PRIN) grant no. 2017FNZRN3 (M.Z.). We also thank NRF 2018R1A5A2025079 Korean Ministry of Science and ICT (J.H.C.). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Ethical Committee approvals for the COVID19 samples use in this study were as follows: (i) protocol no. 141/20; date: 10 April 2020, CEINGE TaskForce Covid19; Azienda Ospedaliera Universitaria Federico II, Direzione Sanitaria, protocol no. 000576 of 10 April 2020; (ii) protocol no. 157/20; date: 22 April 2020, GENECOVID, with the experimental procedures for the use of SAR-CoV-2 in a biosafety level 3 (BSL3) laboratory were authorized by Ministero della Sanita and Dipartimento Di Medicina Molecolare e Biotecnologie Mediche, University degli Studi di Napoli Federico II and Azienda Ospedaliera Universitaria Federico II, Direzione Sanitaria protocol no. 0007133 of 08 May 2020; (iii) protocol no. 18/20; date: 10 June 2020, Genetics CEINGE TaskForce Covid19; Azienda Ospedaliera Universitaria Federico II, Direzione Sanitaria protocol no. 000576 of 10 April 2020. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Prof. Massimo Zollo (massimo.zollo@unina.it). This study did not generate new unique reagents. Gene expression (RNAseq) data are deposited on RNA data bank at EBI (https://www.ebi.ac.uk), July 11. 2022 (code: E-MTAB-11973). Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.