Exploring new catechin derivatives as SARS-CoV-2 Mpro inhibitors from tea by molecular networking, surface plasma resonance, enzyme inhibition, induced fit docking, and metadynamics simulations.

SARS-CoV-2 Mpro (Mpro) is the critical cysteine protease in coronavirus viral replication. Tea polyphenols are effective Mpro inhibitors. Therefore, we aim to isolate and synthesize more novel tea polyphenols from Zhenghedabai (ZHDB) white tea methanol-water (MW) extracts that might inhibit COVID-19. Through molecular networking, 33 compounds were identified and divided into 5 clusters. Further, natural products molecular network (MN) analysis showed that MN1 has new phenylpropanoid-substituted ester-catechin (PSEC), and MN5 has the important basic compound type hydroxycinnamoylcatechins (HCCs). Thus, a new PSEC (1, PSEC636) was isolated, which can be further detected in 14 green tea samples. A series of HCCs were synthesized (2-6), including three new acetylated HCCs (3-5). Then we used surface plasmon resonance (SPR) to analyze the equilibrium dissociation constants (KD) for the interaction of 12 catechins and Mpro. The KD values of PSEC636 (1), EGC-C (2), and EC-CDA (3) were 2.25, 2.81, and 2.44 µM, respectively. Moreover, compounds 1, 2, and 3 showed the potential Mpro inhibition with IC50 5.95 ± 0.17, 9.09 ± 0.22, and 23.10 ± 0.69 µM, respectively. Further, we used induced fit docking (IFD), binding pose metadynamics (BPMD), and molecular dynamics (MD) to explore the stable binding pose of Mpro-1, showing that 1 could tightly bond with the amino acid residues THR26, HIS41, CYS44, TYR54, GLU166, and ASP187. The computer modeling studies reveal that the ester, acetyl, and pyrogallol groups could improve inhibitory activity. Our research suggests that these catechins are effective Mpro inhibitors, and might be developed as therapeutics against COVID-19.

Robust humoral and cellular immune responses in long-term convalescent COVID-19 individuals following one-dose SARS-CoV-2 inactivated vaccination

COVID-19, caused by SARS-CoV-2, has resulted in hundreds of millions of infections and millions of deaths worldwide. Preliminary results exhibited excellent efficacy of SARS-CoV-2 vaccine in preventing hospitalization and severe disease. However, data on inactivated vaccine-induced immune responses of naturally infected patients are limited. Here, we characterized SARS-CoV-2 RBD-specific IgG (anti-S-RBD IgG) and neutralizing antibodies (NAbs) against SARS-CoV-2 wild type and variants of concerns (VOCs), as well as RBD-specific IgG-secreting B cells and antigen-specific T cells respectively in 51 SARS-CoV-2 recovered subjects and 63 healthy individuals. In SARS-CoV-2 recovered patients, a single dose vaccine is sufficient to reactivate robust anti-S-RBD IgG and NAbs. The neutralizing capacity against VOCs increased significantly post-vaccination no matter healthy individuals or SARS-CoV-2 recovered patients. In addition, RBD-specific IgG-secreting B cells in SARS-CoV-2 recovered patients were significantly higher than that in healthy vaccine recipients. After the vaccine booster, the frequencies of specific IFN-γ+ CD4+ T cell, IL-2+ CD4+ T cell, and TNF-α+ CD4+ T cell responses were significantly increased in SARS-CoV-2 recovered patients. Our data highlighted the safety and utility of SARS-CoV-2 inactivated vaccine and demonstrated that robust humoral and cellular immune response can be reactivated by one-dose inactivated vaccine in SARS-CoV-2 recovered patients.

Discovery of honokiol thioethers containing 1,3,4-oxadiazole moieties as potential α-glucosidase and SARS-CoV-2 entry inhibitors.

Honokiol, isolated from a traditional Chinese medicine (TCM) Magnolia officinalis, is a biphenolic compound with several biological activities. To improve and broaden its biological activity, herein, two series of honokiol thioethers bearing 1,3,4-oxadiazole moieties were prepared and assessed for their α-glucosidase and SARS-CoV-2 entry inhibitory activities. Among all the honokiol thioethers, compound 7l exhibited the strongest α-glucosidase inhibitory effect with an IC50 value of 18.9 ± 2.3 µM, which was superior to the reference drug acarbose (IC50 = 24.4 ± 0.3 µM). Some interesting results of structure-activity relationships (SARs) have also been discussed. Enzyme kinetic study demonstrated that 7l was a noncompetitive α-glucosidase inhibitor, which was further supported by the results of molecular docking. Moreover, honokiol thioethers 7e, 9a, 9e, and 9r exhibited potent antiviral activity against SARS-CoV-2 pseudovirus entering into HEK-293 T-ACE2h. Especially 9a displayed the strongest inhibitory activity against SARS-CoV-2 pseudovirus entry with an IC50 value of 16.96 ± 2.45 µM, which was lower than the positive control Evans blue (21.98 ± 1.98 µM). Biolayer interferometry (BLI) binding and docking studies suggested that 9a and 9r may effectively block the binding of SARS-CoV-2 to the host ACE2 receptor through dual recognition of SARS-CoV-2 spike RBD and human ACE2. Additionally, the potent honokiol thioethers 7l, 9a, and 9r displayed relatively no cytotoxicity to normal cells (LO2). These findings will provide a theoretical basis for the discovery of honokiol derivatives as potential both α-glucosidase and SARS-CoV-2 entry inhibitors.

Synthesis and Biological Evaluation of Honokiol Derivatives Bearing 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones as Potential Viral Entry Inhibitors against SARS-CoV-2.

The 2019 coronavirus disease (COVID-19) caused by SARS-CoV-2 virus infection has posed a serious danger to global health and the economy. However, SARS-CoV-2 medications that are specific and effective are still being developed. Honokiol is a bioactive component from Magnoliae officinalis Cortex with damp-drying effect. To develop new potent antiviral molecules, a series of novel honokiol analogues were synthesized by introducing various 3-((5-phenyl-1,3,4-oxadiazol-2-yl)methyl)oxazol-2(3H)-ones to its molecule. In a SARS-CoV-2 pseudovirus model, all honokiol derivatives were examined for their antiviral entry activities. As a result, 6a and 6p demonstrated antiviral entry effect with IC50 values of 29.23 and 9.82 µM, respectively. However, the parental honokiol had a very weak antiviral activity with an IC50 value more than 50 µM. A biolayer interfero-metry (BLI) binding assay and molecular docking study revealed that 6p binds to human ACE2 protein with higher binding affinity and lower binding energy than the parental honokiol. A competitive ELISA assay confirmed the inhibitory effect of 6p on SARS-CoV-2 spike RBD's binding with ACE2. Importantly, 6a and 6p (TC50 > 100 µM) also had higher biological safety for host cells than honokiol (TC50 of 48.23 µM). This research may contribute to the discovery of potential viral entrance inhibitors for the SARS-CoV-2 virus, although 6p's antiviral efficacy needs to be validated on SARS-CoV-2 viral strains in a biosafety level 3 facility.

Impaired cytotoxic cd8⁺ T cell response in elderly COVID-19 patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a T cell response that most likely contributes to virus control in COVID-19 patients but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8⁺ T cells, but not CD4⁺ T cells, characterized by the simultaneous production of granzyme A and B as well as perforin within different effector CD8⁺ T cell subsets. PD-1- expressing CD8⁺ T cells also produced cytotoxic molecules during acute infection, indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years, the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8⁺ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provide valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development. IMPORTANCE Cytotoxic T cells are responsible for the elimination of infected cells and are key players in the control of viruses. CD8⁺ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8⁺ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8⁺ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group than in younger patients.