BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Design, synthesis and structure-activity relationship optimization of phenanthridine derivatives as new anti-vitiligo compounds.

Humans have been suffering from vitiligo for a long time. Target vitiligo drugs have yet been approved. Activation of Wnt/ß-catenin signalling has potential in the therapeutic use of vitiligo, so exploring new drugs that specifically directly activate Wnt is worthwhile to obtain new anti-vitiligo agents. In this work, two portions design and synthesis were put into effect. firstly, 17 phenanthridine derivatives with C-4 substitutes were designed and synthesized, which compounds 4, 6, 12, 13 served as H-acceptor with protein showed enhance melanogenesis activity; Secondly, 7 hybrid new scaffolds of compounds were designed and synthesized, scaffold hopping compound 36 that aromatic benzene was replaced pyrazole on ring C showed enhance melanogenesis and tyrosinase activity; The last and most important, a comprehensive optimization and SARs of compound 36 were carried out, compounds 41 and 43 shared phenolic hydroxyl or 3-methyl-pyridine substitutes at C-7 position remarkably improved the capacity of melanogenesis and tyrosinase activity. Compound 43 were identified as new anti-vitiligo agents that specifically activate the Wnt/ß-catenin signalling pathway by targeting Axin. Structure-activity relationship analysis implied that H-acceptor substitutions at the C-4 position and phenolic hydroxyl or pyridine substitutions at the C-7 position would improve the activities of the compounds. These findings reveal a new therapeutic strategy for vitiligo, and compounds 41 and 43 may represent potential compounds for vitiligo treatment.

Phenanthridine Derivative Host Heat Shock Cognate 70 Down-Regulators as Porcine Epidemic Diarrhea Virus Inhibitors.

Porcine epidemic diarrhea virus (PEDV) has become increasingly problematic around the world, not only for its hazards to livestock but also due to the possibility that it is a zoonotic disease. Although vaccine therapy has made some progress toward PEDV control, additional effective therapeutic strategies against PEDV are needed, such as the development of chemotherapeutic agents. The aim of this work was to identify novel anti-PEDV agents by designing and synthesizing a series of phenanthridine derivatives. Among them, three compounds (compounds 1, 2, and 4) were identified as potent anti-PEDV agents exhibiting suppression of host cell heat shock cognate 70 (Hsc70) expression. Mechanism studies revealed that host Hsc70 is involved in the replication of PEDV, and its expression can be suppressed by destabilization of the mRNA, resulting in inhibition of PEDV replication. Activity against PEDV in vivo in PEDV-infected piglets suggested that phenanthridine derivatives are the first host-acting potential anti-PEDV agents.

Aphananoid A is an Anti-Inflammatory Limonoid with a New 5/6/5 Fused Ring Featuring a C24 Carbon Skeleton from Aphanamixis polystachya.

Aphananoid A, a limonoid which features a rare C24 appendage and new 5/6/5 fused-ring framework, was obtained from Aphanamixis polystachya. The planar structure as well as the absolute configuration was identified based on extensive spectroscopic analysis and electronic circular dichroism calculations. The biogenetic pathway of aphananoid A was also speculated, which arises from the triterpene by the 3,4-seco-7,8-seco-6,8 cyclo-7,30-decarbon key pattern. In addition, bioassays indicated that aphananoid A inhibited NO production in the RAW264.7 cell line (46.80 ± 1.93%).

Design, synthesis and structure-activity relationship optimization of phenanthridine derivatives as new Wnt/ß-catenin signalling pathway agonists.

Phenanthridine derivativeHLY78 has previously been identified as the first Wnt/ß-catenin signalling pathway agonist that targets the DAX domain of axin. However, due to the relatively weak activation on the Wnt/ß-catenin signalling pathway, HLY78 is insufficient for further pharmacological study. Herein, the structural optimization of HLY78 and analyses of the structure-activity relationships (SARs) of HLY78-derived phenanthridine derivatives as agonists of the Wnt/ß-catenin signalling pathway are presented. In this work, 36 derivatives were designed and synthesized with some derivatives exhibiting stronger Wnt activity than the activity of HLY78. In particular, one of them, 8-((1,3-dimethy-pyrazol-5-yl)methoxy)-5-ethyl-4-methyl-5,6-dihydro-phenanthridin-9-ol, exhibited strong Wnt active activity and is 10 times more potent than HLY78. The following SAR analysis suggests that a pyrazole group, especially at the C-8 position, is important for Wnt activation; a methyl group at the C-4position seems to be more beneficial for Wnt activation than ethyl; and oxidation of the C-6 position reduces the Wnt activation.

Design, Synthesis, and Structural Optimization of Lycorine-Derived Phenanthridine Derivatives as Wnt/ß-Catenin Signaling Pathway Agonists.

Lycorine is a benzylphenethylamine-type alkaloid member of the Amaryllidaceae family. A lycorine derivative, HLY78, was previously identified as a new Wnt/ß-catenin signaling pathway agonist that targets the DAX domain of axin. Herein, the structural optimization of HLY78 and analyses of the structure-activity relationships of lycorine-derived phenanthridine derivatives as agonists of the Wnt/ß-catenin signaling pathway are presented. This research suggests that triazole groups are important pharmacophores for Wnt activation; triazole groups at C-8 and C-9 of phenanthridine compounds markedly enhanced Wnt activation. A C-11-C-12 single bond is also important for Wnt activation. On the basis of these findings, two Wnt agonists were designed and synthesized. The results for these agonists indicated that the combination of a 4-ethyldihydrophenanthridine skeleton and a triazole substituent improves Wnt activation. These compounds may be useful in further pharmacological or biological studies.

Limonoids from Aphanamixis polystachya and their antifeedant activity.

Eight new aphanamixoid-type aphanamixoids (C-J, 1-8) and six new prieurianin-type limonoids, aphanamixoids K-P (9-14), along with 10 known terpenoids were isolated from Aphanamixis polystachya, and their structures were established by spectroscopic data analysis. Among the new limonoids, 13 compounds exhibited antifeedant activity against the generalist Helicoverpa armigera, a plant-feeding insect, at various concentration levels. In particular, compounds 1, 4, and 5 showed potent activities with EC50 values of 0.017, 0.008, and 0.012 µmol/cm(2), respectively. On the basis of a preliminary structure-activity relationship analysis, some potential active sites in the aphanamixoid-type limonoid molecules are proposed.

CLC-3 regulates TGF-ß/smad signaling pathway to inhibit the process of fibrosis in hypertrophic scar.

Objective: To study the role and mechanism of chloride channel-3 (ClC-3) in the formation of hypertrophic scar by constructing ClC-3 interference vectors and examining their effects on human hypertrophic scar fibroblasts (HSFB). Methods: Human HSFB and human normal skin fibroblasts (NSFB) were used in this study, and ClC-3 interference vectors were constructed to transfect cells. ClC-3 inhibitors NPPB and Tamoxifen were used to treat cells. Cell migration and the expression of TGF-ß/Smad, CollagenⅠ,CollagenⅢ were examined to explore the role of ClC-3 in the formation of hypertrophic scar. Results: Compared with the normal skin tissue, the positive expression of ClC-3 and TGF-ß in the scar tissue was significantly increased. The relative expression of ClC-3 and TGF-ß1 in HSFB cells was higher than that in NSFB cells. Interfering with the expression of CLC-3 can inhibit the migration of HSFB cells and the expression of TGF- ß/Smad, CollagenⅠ/Ⅲ. The experiment of HSFB cells treated by CLC-3 inhibitors can also obtain similar results. Conclusion: Inhibiting CLC-3 can reduce the formation of hypertrophic scars.

Evaluation of anti-HCV activity and SAR study of (+)-lycoricidine through targeting of host heat-stress cognate 70 (Hsc70).

The anti hepatitis C virus (HCV) activity of (+)-lycoricidine (1) was evaluated for the first time in this letter, yielding an EC50 value of 0.55 nmol/mL and an selection index (SI) value of 12.72. Further studies indicated that 1 induced this effect by down-regulating host heat-stress cognate 70 (Hsc70) expression. In addition, 20 derivatives were designed and synthesised to investigate the basic structure-activity relationship (SAR) of the title compound. Several of these derivatives exhibit a good inhibition of HCV, such as compound 3 (EC50=0.68 nmol/mL, SI=33.86), compound 2d (EC50=15 nmol/mL, SI=12) and compound 5 (EC50=33 nmol/mL, SI >10.91). Meanwhile, the experimental data suggest that the modification of certain groups of (+)-lycoricidine can reduce the cytotoxicity of the compounds.

Establishment of a Colloidal Gold Immunochromatographic Method for the Detection of Cypermethrin in Tobacco.

In this study, the establishment of a colloidal gold immunochromatographic method for the detection of cypermethrin in tobacco was achieved by using colloidal gold immunochromatography: strong specificity and high sensitivity of cypermethrin semi-antigens and encapsulants were prepared during the study. The best colloidal gold solution was prepared by spectrophotometer and transmission electron microscope screening; the preparation process of gold-labeled antibodies was optimized, and finally the product of colloidal gold rapid detection test strips for cypermethrin was developed. The results of technical parameters and detection indexes showed that the detection limit of cypermethrin in tobacco was 1 mg/kg, and there was no cross-reaction with bifenthrin, cypermethrin, cyfluthrin and phenothrin, and the detection results of 30 tobacco samples were consistent with those of gas chromatography.

Development of Colloidal Gold Test Strips for the Detection of Oxamyl Residues in Tobacco.

In this study, monoclonal antibodies against oxamyl were prepared, and colloidal gold immunochromatography was used to design a rapid test strip product for the detection of oxamyl in tobacco with high specificity, accuracy and stability without cross-reactivity to commonly used tobacco fungicides based on the optimization of conditions such as pH value of diluent, diluent dosage, concentration of antibody marker, type of confining solution and complex solution. 5 The results of five samples of post-harvest ready-to-bake tobacco and first-harvest tobacco were consistent with the gas chromatographic method, which proved the reliability of the test strips. The limits of detection for the post-harvest and first-harvest tobacco samples were 0.1 mg/kg, and the test strips developed in this study are suitable for mass testing in tobacco laboratories with good application prospects because of their short detection time, simple pre-treatment and detection methods.

Development of Colloidal Gold Test Strips for the Detection of Triadimenol Residues in Tobacco.

The rapid and accurate determination of triadimenol residues is of great significance. In this study, based on the advantages of high efficiency, rapidity, reliability, simplicity and low cost of immunology, a test strip product for the rapid detection of triadimenol residues in tobacco was designed based on the optimization of conditions such as pH and dosage of diluent, concentration of antibody stock solution, type of confining solution and complex solution, with high specificity, accuracy and The results of 20 samples of fresh and first roasted tobacco were all consistent with the method of gas chromatography, which proved the reliability of the test strips. The detection limit for fresh and roasted tobacco was 5 mg/kg, and the test strips developed in this study are suitable for mass testing of tobacco samples in tobacco-related laboratories because of their short detection time, simple pre-treatment and detection methods, and good application prospects.

Tobacco microbial screening and application in improving the quality of tobacco in different physical states.

The first-cured tobacco contains macromolecular substances with negative impacts on tobacco products quality, and must be aged and fermented to mitigate their effects on the tobacco products quality. However, the natural fermentation takes a longer cycle with large coverage area and low economic efficiency. Microbial fermentation is a method to improve tobacco quality. The change of chemical composition of tobacco during the fermentation is often correlated with shapes of tobacco. This study aimed to investigate the effects of tobacco microorganisms on the quality of different shapes of tobacco. Specifically, Bacillus subtilis B1 and Cytobacillus oceanisediminis C4 with high protease, amylase, and cellulase were isolated from the first-cured tobacco, followed by using them for solid-state fermentation of tobacco powder (TP) and tobacco leaves (TL). Results showed that strains B1 and C4 could significantly improve the sensory quality of TP, enabling it to outperform TL in overall texture and skeleton of tobacco products during cigarette smoking. Compared with the control, microbial fermentation could increase reducing sugar; regulate protein, starch, and cellulose, reduce nicotine, improve total aroma substances, and enable the surface of fermented TP and TL to be more loose, wrinkled, and porous. Microbial community analysis indicated that strains B1 and C4 could change the native structure of microbial community in TP and TL. LEfSe analysis revealed that the potential key biomarkers in TP and TL were Bacilli, Pseudonocardia, Pantoea, and Jeotgalicoccus, which may have cooperative effects with other microbial taxa in improving tobacco quality. This study provides a theoretical basis for improving tobacco fermentation process for better cigarettes quality.

Two new cytotoxic cycloartane triterpenoids from Aphanamixis polystachya (Wall.) R. N. Parker.

Two new cycloartane triterpenoids, (24 R)-cycloartane-3ß,24,25,30-tetrol (1) and (24 R)-24,25,30-trihydroxy-9,19-cycloartane-3-one (2), along with three known compounds (3-5) were isolated from leaves and twigs of Aphanamixis polystachya. The new compounds were elucidated based on comprehensive spectroscopic analysis, including 1 D, 2 D NMR and HREIMS. The in vitro cytotoxic activities evaluation of five human cancer cell lines revealed that compound 1 exhibited cytotoxic activity on all of tested human cancer cell lines, while compound 2 only had specific activity on SMMC-7721 cell line.

Novel nucleocapsid protein-targeting phenanthridine inhibitors of SARS-CoV-2.

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unprecedented in human history. As a major structural protein, nucleocapsid protein (NPro) is critical to the replication of SARS-CoV-2. In this work, 17 NPro-targeting phenanthridine derivatives were rationally designed and synthesized, based on the crystal structure of NPro. Most of these compounds can interact with SARS-CoV-2 NPro tightly and inhibit the replication of SARS-CoV-2 in vitro. Compounds 12 and 16 exhibited the most potent anti-viral activities with 50% effective concentration values of 3.69 and 2.18 µM, respectively. Furthermore, site-directed mutagenesis of NPro and Surface Plasmon Resonance (SPR) assays revealed that 12 and 16 target N-terminal domain (NTD) of NPro by binding to Tyr109. This work found two potent anti-SARS-CoV-2 bioactive compounds and also indicated that SARS-CoV-2 NPro-NTD can be a target for new anti-virus agents.

[Effects of combined natural hirudin and hyperbaric oxygen therapy on survival of transplanted random-pattern skin flap in rats].

Objective: To investigate the effect of natural hirudin combined with hyperbaric oxygen therapy on the survival of transplanted random-pattern skin flap in rats. Methods: A random-pattern skin flap in size of 10.0 cm×2.5 cm was elevated on the dorsum of 72 Sprague Dawley rats. Then the 72 rats were randomly divided into 4 groups ( n=18) according to the therapy method. At immediate and within 4 days after operation, the rats were treated with normal saline injection in control group, normal saline injection combined with hyperbaric oxygen treatment in hyperbaric oxygen group, the natural hirudin injection in natural hirudin group, and the natural hirudin injection combined with hyperbaric oxygen treatment in combined group. The flap survival was observed after operation, and survival rate was evaluated at 6 days after operation. The skin samples were collected for histological analysis, microvessel density (MVD) measurement, and evaluation of tumor necrosis factor α (TNF-α) expression level by the immunohistochemical staining at 2 and 4 days after operation. Results: Partial necrosis occurred in each group after operation, and the flap in combined group had the best survival. The survival rate of flap was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, and in combined group than in hyperbaric oxygen group and natural hirudin group ( P<0.05). There was no significant difference between hyperbaric oxygen group and natural hirudin group ( P>0.05). At 2 days, more microvascular structure was observed in hyperbaric oxygen group, natural hirudin group, and combined group in comparison with control group; while plenty of inflammatory cells infiltration in all groups. At 4 days, the hyperbaric oxygen group, natural hirudin group, and the combined group still showed more angiogenesis. Meanwhile, there was still infiltration of inflammatory cells in control group, inflammatory cells in the other groups were significantly reduced when compared with at 2 days. At 2 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group ( P<0.05); the expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group ( P<0.05). There was no significant difference in above indexes between hyperbaric oxygen group, natural hirudin group, and combined group ( P>0.05). At 4 days, the MVD was significantly higher in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in natural hirudin group and combined group than in hyperbaric oxygen group ( P<0.05). The expression of TNF-α was significantly lower in hyperbaric oxygen group, natural hirudin group, and combined group than that in control group, in combined group than in natural hirudin group and hyperbaric oxygen group ( P<0.05). Conclusion: Hyperbaric oxygen and natural hirudin therapy after random-pattern skin flap transplantation can improve the survival of flaps. Moreover, combined therapy is seen to exhibit significant synergistic effect. This effect maybe related to promotion of angiogenesis and the reduction of inflammation response.

Efficient synthesis of new phenanthridine Wnt/ß-catenin signaling pathway agonists.

Previously, HLY78, a lycorine derivative, was identified as the first Wnt/ß-catenin signaling agonist through binding to the DAX domain of Axin, a scaffold of Wnt/ß-catenin complex. In this study, to obtain more potent Wnt/ß-catenin agonist, the structure optimization of HLY78 was carried out by design and synthesis of six phenanthridine derivatives, which afforded five active ones. In particular, 8,9-bis((1,3-dimethyl-1H-pyrazol)methoxy)-5-ethyl-4-methyl-5,6-dihydrophenanthridine showed the most potent activity (0.15/µM) that was increased nearly 30 times as that of the lead HLY78. These compounds may be valuable in future pharmacological or biological studies.

Design, Synthesis and Structure-Activity Relationship Optimization of Lycorine Derivatives for HCV Inhibition.

Lycorine is reported to be a multifunctional compound. We previously showed that lycorine is an HCV inhibitor with strong activity. Further research on the antivirus mechanism indicated that lycorine does not affect the enzymes that are indispensable to HCV replication but suppresses the expression of Hsc70 in the host cell to limit HCV replication. However, due to the cytotoxicity and apoptosis induction of lycorine, lycorine is unsafe to be a anti-HCV agent for clinical application. As a result of increasing interest, its structure was optimized for the first time and a novel series of lycorine derivatives was synthesized, all of which lost their cytotoxicity to different degrees. Structure-activity analysis of these compounds revealed that disubstitution on the free hydroxyl groups at C1 and C2 and/or degradation of the benzodioxole group would markedly reduce the cytotoxicity. Furthermore, an α, ß-unsaturated ketone would improve the HCV inhibitory activity of lycorine. The C3-C4 double bond is crucial to the anti-HCV activity because hydrogenation of this double bond clearly weakened HCV inhibition.

Lycorine-derived phenanthridine downregulators of host Hsc70 as potential hepatitis C virus inhibitors.

BACKGROUND: A new series of potential phenanthridine hepatitis C virus (HCV) inhibitors which work by suppressing Hsc70 expression in the host cell was designed and synthesized from lycorine. RESULTS: Thirty-one new potential phenanthridine HCV inhibitors were synthesized and five of these compounds exhibited good anti-HCV activity and these inhibitors probably inhibit HCV by downregulating the host Hsc70 expression. Structure-activity analysis of these compounds revealed that the double bond between C-11 and C-12 and the substituents at C-8 and C-9 are important for their activity against HCV. CONCLUSION: Suppression of Hsc70 expression in the host cell to limit HCV replication is a potential anti-HCV strategy. Phenanthridines are probably the HCV inhibitors with this mode of action.