Identification of bis-benzylisoquinoline alkaloids as SARS-CoV-2 entry inhibitors from a library of natural products in vitro

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major public health issue. To screen for antiviral drugs for COVID-19 treatment, we constructed a SARS-CoV-2 spike (S) pseudovirus system using an HIV-1-based lentiviral vector with a luciferase reporter gene to screen 188 small potential antiviral compounds. Using this system, we identified nine compounds, specifically, bis-benzylisoquinoline alkaloids, that potently inhibited SARS-CoV-2 pseudovirus entry, with EC50 values of 0.1-10 M. Mechanistic studies showed that these compounds, reported as calcium channel blockers (CCBs), inhibited Ca2+-mediated membrane fusion and consequently suppressed coronavirus entry. These candidate drugs showed broad-spectrum efficacy against the entry of several coronavirus pseudotypes (SARS-CoV, MERS-CoV, SARS-CoV-2 [S-D614, S-G614, N501Y.V1 and N501Y.V2]) in different cell lines (293T, Calu-3, and A549). Antiviral tests using native SARS-CoV-2 in Vero E6 cells confirmed that four of the drugs (SC9/cepharanthine, SC161/hernandezine, SC171, and SC185/neferine) reduced cytopathic effect and supernatant viral RNA load. Among them, cepharanthine showed the strongest anti-SARS-CoV-2 activity. Collectively, this study offers new lead compounds for coronavirus antiviral drug discovery.

Longitudinal dynamics of the neutralizing antibody response to SARS-CoV-2 infection

BackgroundCoronavirus disease 2019 (COVID-19) is a global pandemic with no licensed vaccine or specific antiviral agents for therapy. Little is known about the longitudinal dynamics of SARS-CoV-2-specific neutralizing antibodies (NAbs) in COVID-19 patients. MethodsBlood samples (n=173) were collected from 30 COVID-19 patients over a 3-month period after symptom onset and analyzed for SARS-CoV-2-specific NAbs, using the lentiviral pseudotype assay, coincident with the levels of IgG and proinflammatory cytokines. ResultsSARS-CoV-2-specific NAb titers were low for the first 7-10 d after symtom onset and increased after 2-3 weeks. The median peak time for NAbs was 33 d (IQR 24-59 d) after symptom onset. NAb titers in 93.3% (28/30) of the patients declined gradually over the 3-month study period, with a median decrease of 34.8% (IQR 19.6-42.4%). NAb titers increased over time in parallel with the rise in IgG antibody levels, correlating well at week 3 (r = 0.41, p < 0.05). The NAb titers also demonstrated a significant positive correlation with levels of plasma proinflammatory cytokines, including SCF, TRAIL, and M-CSF. ConclusionsThese data provide useful information regarding dynamic changes in NAbs in COVID-19 patients during the acute and convalescent phases.

The D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity and decreases neutralization sensitivity to individual convalescent sera

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein that mediates SARS-CoV-2 entry into host cells is a major target for vaccines and therapeutics. Thus, insights into its sequence variations are key to understanding the infection and antigenicity of SARS-CoV-2. A dominant mutational variant at position 614 of the S protein (aspartate to glycine, D614G mutation) was observed in the SARS-CoV-2 genome sequence obtained from the Nextstrain database. Using a pseudovirus-based assay, we identified that S-D614 and S-G614 protein pseudotyped viruses share a common receptor, human angiotensin-converting enzyme 2 (ACE2), which could be blocked by recombinant ACE2 with the fused Fc region of human IgG1. However, S-D614 and S-G614 protein demonstrated functional differences. First, S-G614 protein could be cleaved by serine protease elastase-2 more efficiently. Second, S-G614 pseudovirus infected 293T-ACE2 cells significantly more efficiently than did the S-D614 pseudovirus, especially in the presence of elastase-2. Third, an elastase inhibitor approved for clinical use blocked elastase-enhanced S-G614 pseudovirus infection. Moreover, 93% (65/70) convalescent sera from patients with COVID-19 could neutralize both S-D614 and S-G614 pseudoviruses with comparable efficiencies, but about 7% (5/70) convalescent sera showed reduced neutralizing activity against the S-G614 pseudovirus. These findings have important implications for SARS-CoV-2 transmission and immune interventions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

The adjuvant effects of high-molecule-weight polysaccharides purified from Antrodia cinnamomea on dendritic cell function and DNA vaccines.

The biological activity of the edible basidiomycete Antrodia cinnamomea (AC) has been studied extensively. Many effects, such as anti-cancer, anti-inflammatory, and antioxidant activities, have been reported from either crude extracts or compounds isolated from AC. However, research addressing the function of AC in enhancing immunity is rare. The aim of the present study is to investigate the active components and the mechanism involved in the immunostimulatory effect of AC. We found that polysaccharides (PS) in the water extract of AC played a major role in dendritic cell (DC) activation, which is a critical leukocyte in initiating immune responses. We further size purified and identified that the high-molecular weight PS fraction (greater than 100 kDa) exhibited the activating effect. The AC high-molecular weight PSs (AC hmwPSs) promoted pro-inflammatory cytokine production by DCs and the maturation of DCs. In addition, DC-induced antigen-specific T cell activation and Th1 differentiation were increased by AC hmwPSs. In studying the molecular mechanism, we confirmed the activation of the MAPK and NF-κB pathways in DCs after AC hmwPSs treatment. Furthermore, we demonstrated that TLR2 and TLR4 are required for the stimulatory activity of AC hmwPSs on DCs. In a mouse tumor model, we demonstrated that AC hmwPSs enhanced the anti-tumor efficacy of the HER-2/neu DNA vaccine by facilitating specific Th1 responses. Thus, we conclude that hmwPSs are the major components of AC that stimulate DCs via the TLR2/TLR4 and NF-κB/MAPK signaling pathways. The AC hmwPSs have potential to be applied as adjuvants.

Phloroglucinols with prooxidant activity from Garcinia subelliptica.

A new phloroglucinol, garcinielliptone HF ( 1), possessing an unprecedented skeleton, and the tautomeric pair of garcinielliptone FC ( 2/ 2a) were isolated from the heartwood and pericarp of Garcinia subelliptica, respectively. Their structures, including relative configurations, were elucidated by means of spectroscopic methods. The ability of compounds 1 and 2/ 2a to induce DNA-cleavage activity was examined using supercoiled plasmid pBR322 DNA. In the presence of Cu(II), compounds 1 and 2/ 2a caused significant breakage of pBR322 DNA. The involvement of H2O2 and O2 (*-), and H2O2, O2 (*-), and OH (*) in 1- and 2/ 2a-mediated scission, respectively, was established by inhibition or no protection of DNA breakage by various oxygen radical scavengers. Thus, in the presence Cu(II), 1 and 2/ 2a may show a prooxidant effect on DNA and induce cell death.

DNA strand-scission by phloroglucinols and lignans from heartwood of Garcinia subelliptica Merr. and Justicia plants.

Five 2,4,6-prenylated phloroglucinols, garcinielliptones HA (1), HB (2), HC (3), HD (4) and HE (5), were isolated from the heartwood of Garcinia subelliptica Merr. Their structures, including relative configurations, were elucidated by means of spectroscopic data analysis. The ability of phloroglucinols, 1-5 and lignans, tuberculatin (8), justicidin A (9), procumbenoside A (10) and ciliatosides A (11) and B (12), isolated from Justicia ciliata and Justicia procumbens, to induce DNA-cleavage activity was examined using pBR322, a supercoiled, covalently closed circular DNA, and it was analyzed by agarose gel electrophoresis. In the presence of Cu (II), compounds 3, 8, 10 and 11 caused significant breakage of supercoiled plasmid pBR322. The products were relaxed circles with no detectable linear forms. In the Cu(II)-mediated DNA damage of 3 and selective compound 8, Cu(I) was shown not to be an essential intermediate by using the Cu(I)-specific sequestering reagent neocuproine.

Antiplatelet prenylflavonoids from Artocarpus communis.

Four flavonoids, dihydroartomunoxanthone (1), artomunoisoxanthone (2), cyclocomunomethonol (3) and artomunoflavanone (4), together with three known compounds, artochamins B (5), D and artocommunol CC (6) were isolated from the cortex of the roots of Artocarpus communis. The structures of 1-4 were determined by spectroscopic methods. The antiplatelet effects of the flavonoids, 1-3, 5 and 6 on human platelet-rich plasma (PRP) were evaluated. Of the compounds tested in human PRP, compounds 1, 5 and 6 showed significant inhibition of secondary aggregation induced by adrenaline. It is concluded that the antiplatelet effect of 1, 5 and 6 is mainly owing to an inhibitory effect on thromboxane formation.

Cytotoxic prenylflavonoids from Artocarpus elasticus.

Five new prenylated flavonoids, artelastoheterol (1), artelasticinol (2), cycloartelastoxanthone (3), artelastoxanthone (4), and cycloartelastoxanthendiol (5), along with five known compounds, were isolated from the root bark of Artocarpus elasticus. The structures of 1-5 were elucidated by spectroscopic methods and through comparison with data reported in the literature. The previously known compound artonol A (6) exhibited cytotoxic activity against the A549 human cancer cell line, with an ED50 value of 1.1 microg/mL.