Atazanavir, Alone or in Combination with Ritonavir, Inhibits SARS-CoV-2 Replication and Proinflammatory Cytokine Production.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.

Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients.

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

Both osteopontin-c and osteopontin-b splicing isoforms exert pro-tumorigenic roles in prostate cancer cells.

BACKGROUND: Alternative splicing of the osteopontin (opn, spp1) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb, and OPNc, which have demonstrated specific roles in different tumor models. This work aims to investigate the roles of each OPN-SI in prostate cancer (PCa) progression by using in vivo and in vitro functional assays. METHODS: The expression levels of OPN-SI in prostate cell lines were analyzed by qRT-PCR. PC-3 was stably transfected with expression vectors containing OPNa, OPNb, and OPNc, as well as empty vector controls. PC-3 cells overexpressing each construct were analyzed for in vivo tumor growth and in relation to different aspects mimicking tumor progression, such as cell proliferation, migration, invasion, and soft agar colony formation. RESULTS: OPN-SI are overexpressed in PCa as compared to non-tumoral prostate cell lines. OPNc and OPNb overexpressing cells significantly activated enhanced xenograft tumor growth and PC-3 proliferation, migration, invasion, and soft agar colony formation, as well as the expression of MMP-2, MMP-9, and VEGF. These isoforms also support sustained proliferative survival. We found that both OPNc and OPNb pro-tumorigenic roles are mainly mediated through PI3K signaling. Inhibition of this pathway by using LY294002 specifically inhibited tumor progression features evoked by OPNc and OPNb overexpression. CONCLUSIONS: Our data provide evidence that both OPNc and OPNb splicing isoforms promote distinct aspects of PCa progression by inducing PI3K signaling. These data give support to strategies aiming to downregulate OPNc and OPNb expression as an approach to inhibit PCa progression.