Biosynthesis of rumbrins and inspiration for discovery of HIV inhibitors.

Investigation on how nature produces natural compounds with chemical and biological diversity at the genetic level offers inspiration for the discovery of new natural products and even their biological targets. The polyketide rumbrin (1) is a lipid peroxide production and calcium accumulation inhibitor, which contains a chlorinated pyrrole moiety that is a rare chemical feature in fungal natural products. Here, we identify the biosynthetic gene cluster (BGC) rum of 1 and its isomer 12E-rumbrin (2) from Auxarthron umbrinum DSM3193, and elucidate their biosynthetic pathway based on heterologous expression, chemical complementation, and isotopic labeling. We show that rumbrins are assembled by a highly reducing polyketide synthase (HRPKS) that uniquely incorporates a proline-derived pyrrolyl-CoA starer unit, and followed by methylation and chlorination. Sequent precursor-directed biosynthesis was able to yield a group of rumbrin analogues. Remarkably, inspired by the presence of a human immunodeficiency virus (HIV)-Nef-associated gene in the rum cluster, we predicted and pharmacologically demonstrated rumbrins as potent inhibitors of HIV at the nanomolar level. This work enriches the recognition of unconventional starter units of fungal PKSs and provides a new strategy for genome mining-guided drug discovery.

5-Iodotubercidin inhibits SARS-CoV-2 RNA synthesis.

Coronavirus disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid global emergence of SARS-CoV-2 highlights the importance and urgency for potential drugs to control the pandemic. The functional importance of RNA-dependent RNA polymerase (RdRp) in the viral life cycle, combined with structural conservation and absence of closely related homologs in humans, makes it an attractive target for designing antiviral drugs. Nucleos(t)ide analogs (NAs) are still the most promising broad-spectrum class of viral RdRp inhibitors. In this study, using our previously developed cell-based SARS-CoV-2 RdRp report system, we screened 134 compounds in the Selleckchemicals NAs library. Four candidate compounds, Fludarabine Phosphate, Fludarabine, 6-Thio-20-Deoxyguanosine (6-Thio-dG), and 5-Iodotubercidin, exhibit remarkable potency in inhibiting SARS-CoV-2 RdRp. Among these four compounds, 5-Iodotubercidin exhibited the strongest inhibition upon SARS-CoV-2 RdRp, and was resistant to viral exoribonuclease activity, thus presenting the best antiviral activity against coronavirus from a different genus. Further study showed that the RdRp inhibitory activity of 5-Iodotubercidin is closely related to its capacity to inhibit adenosine kinase (ADK).

Discovery and optimization of 2-((1H-indol-3-yl)thio)-N-benzyl-acetamides as novel SARS-CoV-2 RdRp inhibitors.

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global pandemic coronavirus disease (COVID-19), but no specific antiviral drug has been proven effective for controlling this pandemic to date. In this study, several 2-((indol-3-yl)thio)-N-benzyl-acetamides were identified as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors. After a two-round optimization, a new series of 2-((indol-3-yl)thio)-N-benzyl-acetamides was designed, synthesized, and evaluated for SARS-CoV-2 RdRp inhibitory effect. Compounds 6b2, 6b5, 6c9, 6d2, and 6d5 were identified as potent inhibitors with IC50 values of 3.35 ± 0.21 µM, 4.55 ± 0.2 µM, 1.65 ± 0.05 µM, 3.76 ± 0.79 µM, and 1.11 ± 0.05 µM, respectively; the IC50 of remdesivir (control) was measured as 1.19 ± 0.36 µM. All of the compounds inhibited RNA synthesis by SARS-CoV-2 RdRp. The most potent compound 6d5, which showed a stronger inhibitory activity against the human coronavirus HCoV-OC43 than remdesivir, is a promising candidate for further investigation.

Dengratiols A-D, four new bibenzyl derivatives from Dendrobium gratiossimum.

Dengratiol A (1), an unprecedented bibenzyl derivative bearing a tropolone unit together with three pairs of bibenzyl enantiomers (±)-dengratiols B-D [(±)-2-(±)-4], were isolated from Dendrobium gratiossimum Rchb.f. The resolution of enantiomers was performed with chiral HPLC. Their structures were characterized by extensive spectroscopic data analysis and calculated electronic circular dichroism (ECD). A hypothetical biosynthetic pathway for 1 is proposed. Biological assay revealed that (-)-2 showed moderate antiviral effect against IAV with IC50 value of 6.3 µM, and (±)-2 displayed cytotoxic activities against five human tumor cell lines with IC50 values ranging from 15.5 to 42.5 µM.

Anthraquinone analogues with inhibitory activities against influenza a virus from Polygonatum odoratum.

Three anthraquinone analogues (1-3) were isolated by phytochemical work on EtOAc-soluble ingredients extracted from the roots of Polygonatum odoratum. The structures of all isolates were elucidated by NMR, MS and CD experiments, of which 1 (polygodoquinone A) was identified as a new anthraquinone derivative. Specifically, 1 represents an unusual structure composed of a naphthoquinone derivative linked to an anthraquinone via a C-C bond. 1-3 exhibited remarkable influenza A virus inhibitory activity with IC50 values of 11.4, 11.0, and 2.3 µM, respectively, which were better than ribavirin as the positive control.

New butyrolactone derivatives from the endophytic Fungus Talaromyces sp. CPCC 400783 of Reynoutria japonica Houtt.

Six new butyrolactone derivatives (1, 2a/2b, 3a/3b and 4), together with another two known derivatives (5 and 6) were isolated from the endophytic fungus Talaromyces sp. CPCC 400783. Their structures were established by a combination of spectroscopic analysis, including NMR and HRESIMS. The absolute configurations were elucidated by ECD experiments. Subsequently, compound 1, 3b, 4 and 5 exhibited good inhibitory effect against influenza A/WSN/33 (H1N1) virus with IC50 values of 21.93 ± 1.51, 21.54 ± 3.75, 18.36 ± 2.15 and 23.80 ± 3.05 µM respectively.

Steroidal glycosides, homoisoflavanones and cinnamic acid derivatives from Polygonatum odoratum and their inhibitory effects against influenza A virus.

A phytochemical investigation of Polygonatum odoratum roots led to the isolation of fifteen steroidal glycosides (1-15), three homoisoflavanones (16-18) and four cinnamic acid derivatives (19-22). The structures of all isolated compounds were established mainly by spectroscopic analyses as well as necessary chemical evidence, of which 1-8 (polygodorasides A-G) were identified as new steroidal glycosides. Among the isolates, compounds 7 and 17 showed remarkable in vitro inhibitory effects against influenza A virus with IC50 values of 14.30 and 49.70 µM (positive control ribavirin 28.4 µM).

High-throughput screening of active compounds against human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is one of the predominant pathogens causing lower respiratory tract infection in infants and young children worldwide, whereas there is so far no vaccine or drug against RSV infection for clinical use. In this work, we developed and validated a fluorescence-based high-throughput screening (HTS) assay to identify compounds active against RSV, using RSV-mGFP, a recombinant RSV encoding enhanced green fluorescent protein (EGFP). Thereafter, among 54,800 compounds used for our screen, we obtained 62 compounds active against RSV. Among these hits, azathioprine (AZA) and 6-mercaptopurine (6-MP) were identified as RSV inhibitors with half maximal inhibitory concentration (IC50) values of 6.69 ±â€¯1.41 and 3.13 ±â€¯0.98 µM, respectively. Further experiments revealed that they functioned by targeting virus transcription or/and genome replication. In conclusion, the established HTS assay is suitable to screen anti-RSV compounds, and the screened two hits of AZA and 6-MP, as potential anti-RSV agents targeting RSV genome replication/transcription, are worthy of further investigation on their anti-RSV activity in vivo.

High-Throughput Screening and Identification of Potent Broad-Spectrum Inhibitors of Coronaviruses.

Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressing Renilla luciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVs in vitro We screened 56 hits from the HTS data and validated 36 compounds in vitro using wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVs in vitro at low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration of in vivo real-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.IMPORTANCE Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVs in vitro Additionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

Identification of Triptophenolide from Tripterygium wilfordii as a Pan-antagonist of Androgen Receptor.

A compound, triptophenolide, derived from Tripterygium wilfordii was identified as an antiandrogen. Triptophenolide inhibits the activity of both wild-type and F876L mutant androgen receptors. Triptophenolide exhibits its antiandrogenic activity through competitive binding with androgen in the hormone-binding pocket, decreasing the expression of androgen receptor, and reducing the nuclear translocation of androgen receptor.

2-thio-6-azauridine inhibits Vpu mediated BST-2 degradation.

BACKGROUD: BST-2 is an interferon-induced host restriction factor that inhibits the release of diverse mammalian enveloped viruses from infected cells by physically trapping the newly formed virions onto the host cell surface. Human Immunodeficiency Virus-1 (HIV-1) encodes an accessory protein Vpu that antagonizes BST-2 by down-regulating BST-2 from the cell surface. RESULTS: Using a cell-based ELISA screening system, we have discovered a lead compound, 2-thio-6-azauridine, that restores cell surface BST-2 level in the presence of Vpu. This compound has no effect on the expression of BST-2 and Vpu, but inhibits Vpu-mediated BST-2 down-regulation and exerts no effect on Vpu-induced down-regulation of CD4 or KSHV K5 protein induced BST-2 down-regulation. 2-thio-6-azauridine suppresses HIV-1 production in a BST-2-dependent manner. Further results indicate that 2-thio-6-azauridine does not interrupt the interaction of BST-2 with Vpu and ß-TrCP2, but decreases BST-2 ubiquitination. CONCLUSION: Our study demonstrates the feasibility of using small molecules to target Vpu function and sensitize wild type HIV-1 to BST-2-mediated host restriction.

[Triterpenoid saponins from Bupleurum marginatum var. stenophyllum].

Twelve compounds were obtained by phytochemical investigation of 70% EtOH ( containing 0.5%NH3•H2O )extract of the roots of Bupleurum marginatum var. stenophyllum. Based on comparison of their spectral data, including HR-ESI-MS, ¹H-NMR, ¹³C-NMR data, with those of the literature, their structures were elucidated as saikosaponin b2 (1), saikosaponin a(2), saikosaponin b1(3), saikosaponin d (4), hydroxysaikosaponin a (5), saikosaponin b3 (6), saikosaponin c(7),saikosaponin i (8), saikosaponin f (9), chikusaikosides Ⅱ(10), saikosaponin s (11), and saikosaponin I(12). All compounds belong to olean-type triterpenoid saponin and compounds 1, 3, 5, 8-9,11, and 12 were isolated from this plant for the first time. At a concentration of 20 µmol•L⁻¹, compounds 2, 4, 6, 8, 11 and 12 showed strong inhibition activity against influenza virus WSN33 with the inhibition rate of 91.3%,88.6%,53.4%,61.3%,77.3% and 57.4%,respectively.

[Establishment and application of a drug screening model for anti-prostate cancer agents targeting androgen receptor].

Androgen receptor(AR) plays an important role in the maintenance of prostate function and development of prostate cancer. AR is the key target in the therapy of prostate cancer. In this study, a cell-based screening assay was established by dual-luciferase reporter system to analyze the activity of AR. In the screening assay, we detected the anti-prostate cancer activities of rhodiola root extract, wild kiwifruit root extract and tripterygium wilfordii root extract, which may provide a new strategy for the treatment of prostate cancer.

[Development and Application of An Assay for High-throughput Antiviral Compounds Screening against Alphaviruses].

To establish a cell-based rapid luciferase suppression assay for high-throughput screening (HTS) anti-alphaviruses compounds screening, which could cause viral encephalitis, raise the social issues associated directly with public health and huge economic burden to the society. The Gaussia luciferase assay system was used for HTS model for identifying inhibitors of labeled virus XJ160-GLUC. The decreased 50% GLUC activity inhibition ratio was deemed to be the screening positive index. The reaction system in this model was optimized, and the reliability of the model was evaluated. For HTS model's optimization, cells were infected with XJ160-GLUC at an MOI of 0.025 PFU/cell. The supernatant treated with compounds 48h were collected for GLUC expression detection. In the model, Z' factor was up to 0.71, demonstrating that HTS assay for identifying inhibitors that target all aspects of the viral life cycle of XJ160-GLUC was stable and reliable. After screening 8080 compounds (five-in-one), 341 positive samples were selected, and the positive rate was 4.2% with a cutoff at 50% inhibition. Then 1705 compounds were screened subsequently and the positive rate was 1.1% with obtaining 19 positive compounds. These results will lay the foundation for finding the anti-alphaviruses' drug targets.

[Establishment of drug screening assay and pharmacodynamic evaluation method targeting influenza RNA polymerase].

Influenza virus RNA-dependent RNA polymerase (RdRP) is essential for replication and expression of influenza virus genome. Viral genomic sequences encoding RdRP are highly conservative, thus making it a potential anti-influenza drug target. A cell-based influenza RdRP inhibitor screening assay was established by a luciferase reporter system to analyze the activity of RdRP. Specificity study and statistic analysis showed that the screening assay is sensitive and reproducible.