Identification of SARS-CoV-2 entry inhibitors among already approved drugs.

To discover effective drugs for COVID-19 treatment amongst already clinically approved drugs, we developed a high throughput screening assay for SARS-CoV-2 virus entry inhibitors using SARS2-S pseudotyped virus. An approved drug library of 1800 small molecular drugs was screened for SARS2 entry inhibitors and 15 active drugs were identified as specific SARS2-S pseudovirus entry inhibitors. Antiviral tests using native SARS-CoV-2 virus in Vero E6 cells confirmed that 7 of these drugs (clemastine, amiodarone, trimeprazine, bosutinib, toremifene, flupenthixol, and azelastine) significantly inhibited SARS2 replication, reducing supernatant viral RNA load with a promising level of activity. Three of the drugs were classified as histamine receptor antagonists with clemastine showing the strongest anti-SARS2 activity (EC50 = 0.95 ± 0.83 µM). Our work suggests that these 7 drugs could enter into further in vivo studies and clinical investigations for COVID-19 treatment.

Overview of therapeutic drug research for COVID-19 in China.

Since the outbreak of novel coronavirus pneumonia (COVID-19) in December 2019, more than 2,500,000 people worldwide have been diagnosed with SARS-CoV-2 as of April 22. In response to this epidemic, China has issued seven trial versions of diagnosis and treatment protocol for COVID-19. According to the information that we have collected so far, this article provides an overview of potential therapeutic drugs and compounds with much attention, including favipiravir and hydroxychloroquine, as well as traditional Chinese medicine, which have been reported with good clinical treatment effects. Moreover, with further understanding of SARS-CoV-2 virus, new drugs targeting specific SARS-CoV-2 viral components arise and investigations on these novel anti-SARS-CoV-2 agents are also reviewed.

A novel pyridazinone derivative inhibits hepatitis B virus replication by inducing genome-free capsid formation.

Here we first identified a novel pyridazinone derivative, compound 3711, as a nonnucleosidic hepatitis B virus (HBV) inhibitor in a cell model system. 3711 decreased extracellular HBV DNA levels by 50% (50% inhibitory concentration [IC50]) at 1.5 ± 0.2 µM and intracellular DNA levels at 1.9 ± 0.1 µM, which demonstrated antiviral activity at levels far below those associated with toxicity. Both the 3TC/ETV dually resistant L180M/M204I mutant and the adefovir (ADV)-resistant A181T/N236T mutant were as susceptible to 3711 as wild-type HBV. 3711 treatment induced the formation of genome-free capsids, a portion of which migrated faster on 1.8% native agarose gel. The induced genome-free capsids sedimented more slowly in isopycnic CsCl gradient centrifugation without significant morphological changes. 3711 treatment decreased levels of HBV DNA contained in both secreted enveloped virion and naked virus particles in supernatant. 3711 could interfere with capsid formation of the core protein (Cp) assembly domain. A Cp V124W mutant, which strengthens capsid interdimer interactions, recapitulated the effect of 3711 on capsid assembly. Pyridazinone derivative 3711, a novel chemical entity and HBV inhibitor, may provide a new opportunity to combat chronic HBV infection.

Discovering novel anti-HCV compounds with inhibitory activities toward HCV NS3/4A protease.

AIM: To discover novel hepatitis C virus (HCV) inhibitors and elucidate the mechanism of action of the active compounds. METHODS: HCV subgenomic replicon-based luciferase reporter cell line was used to screen 1200 synthetic compounds with novel structures. Huh7.5.1 cell line stably transfected with HCV NS3/4A protease reporter was established to investigate the anti-HCV mechanism of the active compounds. The active compounds were further examined in an in vitro HCV infection assay to confirm their anti-HCV activity. RESULTS: After two-round screening in the anti-HCV replicon assay, some 2,4-diaminoquinazoline derivatives and carboxamide analogues were found to possess anti-HCV replicon activities (the IC50 values were less than 5 µmol/L). Among them, two representative compounds HZ-1157 and LZ-110618-6 inhibited HCV NS3/4A protease with IC50 values of 1.0 and 0.68 µmol/L, respectively. Furthermore, HZ-1157 and LZ-110618-6 inhibited HCV infection in vitro with IC50 values of 0.82 and 0.11 µmol/L, respectively. CONCLUSION: Some 2,4-diaminoquinazoline derivatives and carboxamide analogues have been identified as novel anti-HCV compounds.

Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation.

AIM: To investigate the action of isothiafludine (NZ-4), a derivative of bis-heterocycle tandem pairs from the natural product leucamide A, on the replication cycle of hepatitis B virus (HBV) in vitro and in vivo. METHODS: HBV replication cycle was monitored in HepG2.2.15 cells using qPCR, qRT-PCR, and Southern and Northern blotting. HBV protein expression and capsid assembly were detected using Western blotting and native agarose gel electrophoresis analysis. The interaction of pregenomic RNA (pgRNA) and the core protein was investigated by RNA immunoprecipitation. To evaluate the anti-HBV effect of NZ-4 in vivo, DHBV-infected ducks were orally administered NZ-4 (25, 50 or 100 mg·kg⁻¹·d⁻¹) for 15 d. RESULTS: NZ-4 suppressed intracellular HBV replication in HepG2.2.15 cells with an IC50 value of 1.33 µmol/L, whereas the compound inhibited the cell viability with an IC50 value of 50.4 µmol/L. Furthermore, NZ-4 was active against the replication of various drug-resistant HBV mutants, including 3TC/ETV-dual-resistant and ADV-resistant HBV mutants. NZ-4 (5, 10, 20 µmol/L) concentration-dependently reduced the encapsidated HBV pgRNA, resulting in the assembly of replication-deficient capsids in HepG2.2.15 cells. Oral administration of NZ-4 dose-dependently inhibited DHBV DNA replication in the DHBV-infected ducks. CONCLUSION: NZ-4 inhibits HBV replication by interfering with the interaction between pgRNA and HBcAg in the capsid assembly process, thus increasing the replication-deficient HBV capsids. Such mechanism of action might provide a new therapeutic strategy to combat HBV infection.

Mycophenolic acid derivative 118 improves outcome of skin grafts by suppressing IL-17 production.

AIM: To investigate the effects and underlying mechanisms of 118, a novel derivative of mycophenolic acid, in a murine allogeneic skin graft model. METHODS: Skin grafts were conducted by grafting BALB/c donor tail skin into C57BL/6 skin beds (allograft) or by grafting female C57BL/6 donor tail skin into female C57BL/6 skin beds (syngraft). The mice were treated with the derivative 118 (40 mg·kg(-1)·d(-1), po) for 13 d (3 d before and 10 d after transplantation). Skin grafts, splenocytes and graft-infiltrated lymphocytes were isolated and examined ex vivo. The effects of the derivative 118 on naive CD4(+) T cell differentiation were examined in vitro. RESULTS: Treatment with the derivative 118 dramatically increased the survival rate of murine allogeneic skin grafts. Flow cytometric analysis and H&E staining showed that the derivative significantly decreased inflammatory cell infiltration into the grafts. The levels of the chemokines CXCL1, CXCL2, CCL7, and CCL2 were reduced in the derivative 118-treated grafts. Additionally, the derivative 118 significantly suppressed the IL-17 levels in the grafts but did not affect the differentiation of systemic helper T cells in the murine allogeneic skin graft model. Furthermore, IL-23p19 expression was suppressed in the grafts from the derivative 118-treated group, which might be due to decreases in TLR4 and MyD88 expression. Finally, the derivative 118 did not exert direct influences on helper T cell differentiation in vitro. CONCLUSION: Treatment with the mycophenolic acid derivative 118 improves murine allogeneic skin grafts by decreasing IL-23 expression and suppressing local IL-17 secretion in the grafts, rather than directly inhibiting Th17 differentiation.

Multiplication of defective Ebola virus in a complementary permissive cell line.

In an effort to develop safe and innovative in vitro models for Ebola virus (EBOV) research, we generated a recombinant Ebola virus where the glycoprotein (GP) gene was substituted with the Cre recombinase (Cre) gene by reverse genetics. This defective virus could multiply itself in a complementary permissive cell line, which could express GP and reporter protein upon exogenous Cre existence. The main features of this novel model for Ebola virus are intact viral life cycle, robust virus multiplication and normal virions morphology. The design of this model ensures its safety, excellent stability and maneuverability as a tool for virology research as well as for antiviral agent screening and drug discovery, and such a design could be further adapted to other viruses.

WSS45, a sulfated alpha-D-glucan, strongly interferes with Dengue 2 virus infection in vitro.

AIM: To investigate the mode of action of WSS45, one sulfated derivative of an alpha-D-glucan from the Gastrodia elata Bl, on the multiplication cycle of dengue virus serotype 2 (DV2), including initial infection and intracellular replication. METHODS: Virus multiplication in BHK cells were monitored by qRT-PCR, plaque reduction assay, and flow cytometry. Initial virus infection was dissected into adsorption and penetration steps by converting temperature and treating by acid glycine. Surface bound virions were detected by immunofluorescence staining for Evelope protein. RESULTS: WSS45 effectively inhibited DV2 infection in BHK cells with an EC(50) value of 0.68+/-0.17 microg/mL, mainly interfered with virus adsorption, in a very early stage of the virus cycle. However, WSS45 showed no viricidal effect. Moreover, WSS45 could increase the detaching of virus from cell surface in BHK cell line. CONCLUSION: WSS45 exerted potent inhibitory effect on DV2 through interfering with the interaction between viruses and targeted cells. This activity was related to its molecular size.

Microbial quantitation of colostrum from healthy breastfeeding women and milk from mastitis patients.

BACKGROUND: Colostrum is rich in microbiota. However, the quantity of microorganisms including both opportunistic pathogens and commensal mammary microbiota remains fluctuant during lactation. And once dysbiosis occurred in these microorganisms, a process in which the population of opportunistic pathogens increases while other bacteria, commensal mammary microbiota decrease. Lactation mastitis might occur. There were few literatures of microbiota in Chinese breastfeeding women. So this study aimed to investigate the quantity of microbiota in the colostrum from healthy breastfeeding women and the milk from mastitis patients in China. METHODS: From January to December 2017, a total of 400 milk samples were collected from the bilateral breasts of 200 women (104 healthy women and 86 mastitis patients). Microbial quantitation was done based on the conserved marker gene 16s rRNA for Bifidobacterium, Lactobacillus, Staphylococcus and Streptococcus by Real-time PCR in all milk samples. The bacterial culture of milk from mastitis patients was also performed. RESULTS: In the colostrum, the amounts of Lactobacillus and Bifidobacterium were significantly higher than those of Staphylococcus and Streptococcus (P<0.0001). The amounts of all the detected bacteria in the colostrum were significantly higher than in the milk from mastitis patients (P<0.01). The same results were obtained in patients with bacteria unrelated mastitis (P<0.01). With respect to colostrum samples, the Staphylococcus copies increased and Bifidobacterium copies declined in cases caused by Staphylococcus aureus or Methicillinresistant Staphylococcus aureus, while both the Staphylococcus and Bifidobacterium copies declined in the milk from patients with Staphylococcus epidermidis or Staphylococcus Lentus induced mastitis (P<0.05). CONCLUSIONS: Results of this study reveal a large amount of microbiota in the colostrum, and mammary microbial dysbiosis may be involved in the pathogenesis of lactational mastitis.

1-Phenyl-N-(benzothiazol-2-yl)methanimine derivatives as Middle East respiratory syndrome coronavirus inhibitors.

Middle East respiratory syndrome coronavirus (MERS-CoV) poses a serious threat to human health, and currently there are no effective or specific therapies available to treat it. Herein a series of 1-phenyl-N-(benzothiazol-2-yl)methanimine derivatives with inhibitory activity against MERS-CoV are described. The compound 4f with a 50% inhibition concentration value of 0.09 µM is a promising inhibitor that warrants further evaluation, towards the development of potential anti-MERS-CoV drugs.

Effect of a hepatitis B virus inhibitor, NZ-4, on capsid formation.

During the hepatitis B virus (HBV) life cycle, nucleocapsid assembly is essential for HBV replication. Both RNA reverse transcription and DNA replication occur within the HBV nucleocapsid. HBV nucleocapsid is consisted of core protein (HBcAg), whose carboxy-terminal domain (CTD) contains an Arg-rich domain (ARD). The ARD of HBcAg does contribute to the encapsidation of pregenomic RNA (pgRNA). Previously, we reported a small-molecule, NZ-4, which dramatically reduced the HBV DNA level in an in vitro cell setting. Here, we explore the possible mechanisms by which NZ-4 inhibits HBV function. As an HBV inhibitor, NZ-4 leads to the formation of genome-free capsids, including a new population of capsid that runs faster on agarose gels. NZ-4's activity was dependent on the presence of the ARD I, containing at least one positively charged amino acid. NZ-4 might provide a new option for further development of HBV therapeutics for the treatment of chronic hepatitis B.

Benzimidazole derivative, BM601, a novel inhibitor of hepatitis B virus and HBsAg secretion.

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family. HBsAg, greatly outnumbered mature virion, has been mysterious since the discovery of HBV. A novel benzimidazole derivative, BM601, is identified inhibiting the secretion of HBV virions and HBsAg, with 50% effective concentration of 0.6µM and 1.5µM, as well as 50% cytotoxicity concentration of 24.5µM. It has no effect on transcription, protein production, nucleocapsid formation or intracellular HBV DNA synthesis. Immunofluorescence analysis suggests that BM601 might inhibit virion and HBsAg secretion by interfering surface protein aggregation in trans Golgi apparatus. Furthermore, BM601 does not trigger cellular stress response or affect HBeAg or host protein secretion. We hypothesize that BM601 is a secretion inhibitor functioning at the level of virion and HBsAg secretion pathway.

Discovery and optimization of 2,4-diaminoquinazoline derivatives as a new class of potent dengue virus inhibitors.

The results of a high-throughput screening assay using the DENV-2 replicon showed that the 2,4-diaminoquinazoline derivative 4a has a high dengue virus inhibitory activity (EC(50) = 0.15 µM). A series of 2,4-diaminoquinazoline derivatives based on 4a as a lead compound were synthesized and subjected to structure-antidengue activity relationship studies. Among the series of 2,4-diaminoquinazoline derivative probed, 4o was observed to display both the highest antiviral potency (EC(50) = 2.8 nM, SI > 1000) and an excellent pharmacokinetic profile.

The chemokine receptor antagonist, TAK-779, decreased experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system, without affecting T cell function.

BACKGROUND AND PURPOSE: The C-C chemokine receptor CCR5, and the C-X-C chemokine receptor CXCR3 are involved in the regulation of T cell-mediated immune responses, and in the migration and activation of these cells. To determine whether blockade of these chemokine receptors modulated inflammatory responses in the central nervous sytem (CNS), we investigated the effect of a non-peptide chemokine receptor antagonist, TAK-779, in mice with experimental autoimmune encephalomyelitis (EAE). EXPERIMENTAL APPROACH: EAE was induced by immunization of C57BL/6 mice with myelin oligodendrocyte glycoprotein (MOG) 35-55. TAK-779 was injected s.c. once a day after immunization. Disease incidence and severity (over 3 weeks) were monitored by histopathological evaluation and FACS assay of inflammatory cells infiltrating into the spinal cord, polymerase chain reaction quantification of mRNA expression, assay of T cell proliferation, by [3H]-thymidine incorporation and cytokine production by enzyme-linked immunosorbent assay. KEY RESULTS: Treatment with TAK-779 reduced incidence and severity of EAE. It strongly inhibited migration of CXCR3/CCR5 bearing CD4+, CD8+ and CD11b+ leukocytes to the CNS. TAK-779 did not reduce proliferation of anti-MOG T cells, the production of IFN-gamma by T cells or CXCR3 expression on T cells. In addition, TAK-779 did not affect production of IL-12 by antigen-presenting cells, CCR5 induction on T cells and the potential of MOG-specific T cells to transfer EAE. CONCLUSIONS AND IMPLICATIONS: TAK-779 restricted the development of MOG-induced EAE. This effect involved reduced migration of inflammatory cells into the CNS without affecting responses of anti-MOG T cells or the ability of MOG-specific T cells to transfer EAE.