<i>In vitro</i> antiviral activity of the anti-HCV drugs daclatasvir and sofosbuvir against SARS-CoV-2, the aetiological agent of COVID-19

BackgroundCurrent approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.MethodsSARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir's dose and schedule to maximize the probability of success for COVID-19.ResultsDaclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.ConclusionsDaclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy

The Neuropeptides Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Control HIV-1 Infection in Macrophages Through Activation of Protein Kinases A and C

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are highly similar neuropeptides present in several tissues, endowed with immunoregulatory functions and other systemic effects. We previously reported that both neuropeptides reduce viral production in HIV-1-infected primary macrophages, with the participation of β-chemokines and IL-10, and now we describe molecular mechanisms engaged in this activity. Macrophages exposed to VIP or PACAP before HIV-1 infection showed resistance to viral replication, comparable to that observed when the cells were treated after infection. Also, multiple treatments with a suboptimal dose of VIP or PACAP after macrophage infection resulted in a decline of virus production similar to the inhibition promoted by a single exposure to the optimal inhibitory concentration. Cellular signaling pathways involving cAMP production and activation of protein kinases A and C were critical components of the VIP and PACAP anti-HIV-1 effects. Analysis of the transcription factors and the transcriptional/cell cycle regulators showed that VIP and PACAP induced cAMP response element-binding protein activation, inhibited NF-kB, and reduced Cyclin D1 levels in HIV-1-infected cells. Remarkably, VIP and PACAP promoted G-to-A mutations in the HIV-1 provirus, matching those derived from the activity of the APOBEC family of viral restriction factors, and reduced viral infectivity. In conclusion, our findings strengthen the antiretroviral potential of VIP and PACAP and point to new therapeutic approaches to control the progression of HIV-1 infection

Macrophage Resistance to HIV-1 Infection Is Enhanced by the Neuropeptides VIP and PACAP

<div><p>It is well established that host factors can modulate HIV-1 replication in macrophages, critical cells in the pathogenesis of HIV-1 infection due to their ability to continuously produce virus. The neuropeptides VIP and PACAP induce well-characterized effects on macrophages through binding to the G protein-coupled receptors VPAC1, VPAC2 and PAC1, but their influence on HIV-1 production by these cells has not been established. Here, we describe that VIP and PACAP reduce macrophage production of HIV-1, acting in a synergistic or additive manner to decrease viral growth. Using receptor antagonists, we detected that the HIV-1 inhibition promoted by VIP is dependent on its ligation to VPAC1/2, whereas PACAP decreases HIV-1 growth via activation of the VPAC1/2 and PAC1 receptors. Specific agonists of VPAC2 or PAC1 decrease macrophage production of HIV-1, whereas sole activation of VPAC1 enhances viral growth. However, the combination of specific agonists mimicking the receptor preference of the natural neuropeptides reproduces the ability of VIP and PACAP to increase macrophage resistance to HIV-1 replication. VIP and PACAP up-regulated macrophage secretion of the β-chemokines CCL3 and CCL5 and the cytokine IL-10, whose neutralization reversed the neuropeptide-induced inhibition of HIV-1 replication. Our results suggest that VIP and PACAP and the receptors VPAC2 and PAC1 could be used as targets for developing alternative therapeutic strategies for HIV-1 infection.</p></div

Effects of combined VIP and PACAP treatment on HIV replication

<p>. HIV-1-infected macrophages were simultaneously treated with 1 nM, 5 nM or 10 nM of VIP and PACAP (A, B and C, respectively), and virus replication was measured as above. Data represent means ± SEM of three independent experiments. (D) Equation used to calculate the interaction coefficient of VIP and PACAP at the indicated concentrations, based on the levels of HIV-1 inhibition shown in A, B and C. *<i>p</i>≤.05; **<i>p</i>≤.01; ***<i>p</i>≤.001.</p

VIP and PACAP inhibit HIV-1 replication

<p>. Macrophages were infected with an R5-tropic HIV-1 isolate (Ba-L) and treated once with different concentrations of the neuropeptides, as indicated. Virus replication was measured in the culture supernatants by an HIV-1 p24 ELISA 12-14 days after infection. Data represent means ± SEM of five independent experiments for each peptide. *<i>p</i>≤.05; ***<i>p</i>≤.001.</p

Combined use of receptor agonists reproduces VIP and PACAP effects on HIV-1 replication

<p>. Macrophages were infected with an R5-tropic HIV-1 isolate (Ba-L) and treated with agonists for the VPAC1 (<i>agVPAC1</i> 2.5 nM), VPAC2 (<i>agVPAC2</i> 2.5 nM) or PAC1 (<i>agPAC1</i>, 5 nM) receptors or with VIP (5 nM) and PACAP (5 nM), either alone or in combination, as indicated. Viral replication was measured in the culture supernatants using an HIV-1 p24 ELISA 12–14 days after infection. Viral production in the positive control (HIV-1-infected cells cultured only with medium): 3.0±0.8 ng/mL p24 Ag. **<i>p</i>≤.01; ***<i>p</i>≤.001.</p

Contribution of VIP and PACAP receptors for the neuropeptide-induced inhibition of HIV-1 replication

<p>. Macrophages were infected with an R5-tropic HIV-1 isolate (Ba-L) and treated with culture medium (<i>Medium</i>) or with an antagonist of PAC1 (<i>M65</i>, 50 nM), VPAC1 and VPAC2 (<i>atVIP,</i> 100 nM) or with both antagonists (<i>M65+atVIP</i>) 15 minutes before the addition of VIP (A) or PACAP (B) at 10 nM. Viral replication was measured in the culture supernatants using an HIV-1 p24 ELISA 12-14 days after infection. Data represent means ± SEM of five independent experiments. Virus production in the positive control (HIV-1-infected cells cultured only with medium): 5.8±1.9 ng/mL p24 Ag. The three bars on the right show the virus replication by macrophages exposed only to the antagonists. *<i>p</i>≤.05; **<i>p</i>≤.01; ***<i>p</i>≤.001.</p

β-chemokines and IL-10 are implicated in the VIP- and PACAP-induced inhibition of HIV-1 replication

<p>. Macrophages were infected with an R5-tropic HIV-1 isolate (Ba-L), and after 5 days, were treated with VIP or PACAP plus anti-CCL3, CCL4 and CCL5 antibodies (<i>α-βC)</i> (A), anti-IL-10 receptor antibodies (<i>α-IL10R</i>), or isotype control antibodies. Viral replication was measured in the culture supernatants using an HIV-1 p24 ELISA 12–14 days after infection. Data represent means ± SEM of five (A) or four (B) independent experiments. Virus production in the positive control (HIV-1-infected cells cultured only with medium): (A) 14.8±9.0 ng/mL and (B) 14.5±7.0 ng/mL p24 Ag. *<i>p</i>≤.05; **<i>p</i>≤.01.</p

VIP and PACAP induce CCL3, CCL5 and IL-10 production in macrophages

<p>. Figure shows the production of CCL3 (A), CCL5 (B) and IL-10 (C) by area under the curve (AUC) analysis, which was calculated based on the respective concentrations measured by ELISA (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067701#pone.0067701.s001" target="_blank">Figure S1</a>). Data represent means ± SEM of six (CCL3) and four (CCL5 and IL10) independent experiments. *<i>p</i>≤.05; **<i>p</i>≤.01; ***<i>p</i>≤.001.</p

Specific activity of VIP and PACAP receptors on HIV-1 replication

<p>. Macrophages were infected with an R5-tropic HIV-1 isolate (Ba-L) and treated once with different concentrations of agonists for the VPAC1 (<i>agVPAC1</i>), VPAC2 (<i>agVPAC2</i>) or PAC1 (<i>agPAC1</i>) receptors, as indicated, and viral replication was measured in the culture supernatants using an HIV-1 p24 ELISA 12-14 days after infection. Data represent means ± SEM of four independent experiments. *<i>p</i>≤.05.</p