Template-based modeling and ab-initio docking using CoDock in CAPRI.

Integration of template-based modeling, global sampling and precise scoring is crucial for the development of molecular docking programs with improved accuracy. We combined template-based modeling and ab-initio docking protocol as hybrid docking strategy called CoDock for the docking and scoring experiments of the seventh CAPRI edition. For CAPRI rounds 38-45, we obtained acceptable or better models in the top 10 submissions for eight out of the 16 evaluated targets as predictors, nine out of the 16 targets as scorers. Especially, we submitted acceptable models for all of the evaluated protein-oligosaccharide targets. For the CASP13-CAPRI experiment (round 46), we obtained acceptable or better models in the top 5 submissions for 10 out of the 20 evaluated targets as predictors, 11 out of the 20 targets as scorers. The failed cases for our group were mainly the difficult targets and the protein-peptide systems in CAPRI and CASP13-CAPRI experiments. In summary, this CAPRI edition showed that our hybrid docking strategy can be efficiently adapted to the increasing variety of challenges in the field of molecular interactions.

The selenium-containing drug ebselen potently disrupts LEDGF/p75-HIV-1 integrase interaction by targeting LEDGF/p75.

Lens-epithelium-derived growth-factor (LEDGF/p75)-binding site on HIV-1 integrase (IN), is an attractive target for antiviral chemotherapy. Small-molecule compounds binding to this site are referred as LEDGF-IN inhibitors (LEDGINs). In this study, compound libraries were screened to identify new inhibitors of LEDGF/p75-IN interaction. Ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a reported anti-HIV-1 agent, was identified as a moderate micromolar inhibitor of LEDGF/p75-IN interaction. Ebselen inhibited the interaction by binding to LEDGF/p75 and the ability of ebselen to inhibit the interaction could be reversed by dithiothreitol (DTT). BLI experiment showed that ebselen probably formed selenium-sulphur bonds with reduced thiols in LEDGF/p75. To the best of our knowledge, we showed for the first time that small-molecule compound, ebselen inhibited LEDGF/p75-IN interaction by directly binding to LEDGF/p75. The compound discovered here could be used as probe compounds to design and develop new disrupter of LEDGF/p75-IN interaction.

Utilization of UPLC/Q-TOF-MS-Based Metabolomics and AFLP-Based Marker-Assisted Selection to Facilitate/Assist Conventional Breeding of Polygala tenuifolia.

As one of the most important traditional Chinese medicine, the quality of Polygala tenuifolia is difficult to control and a new method must be established to facilitate/assist the breeding of P. tenuifolia. In this study, UPLC/Q-TOF-MS-based metabolomics analysis was performed to determine the chemical composition and screen metabolite biomarkers according to agronomic traits. A total of 29 compounds and 18 metabolite biomarkers were found. AFLP-based marker-assisted selection (MAS) was used to identify molecular marker bands and screen characteristic bands associated with specific agronomic traits. 184 bands and 76 characteristic AFLP bands were found. The correlation network between compounds and characteristic AFLP bands was built, so we may directly breed certain P. tenuifolia herbs with special agronomic traits (or characteristic AFLP bands), which exhibit specific pharmacological functions depending on the content of the active compounds. The proposed method of metabolomics coupled with MAS could facilitate/assist the breeding of P. tenuifolia.

[Analysis of digital gene expression profiles of the cultivated Polygala tenuifolia in different phenological phases].

The content changes of chemical components in different phenological phase of the cultivated Polygala tenuifolia is one of the important factors for determination of the best harvest time in the production practice. In this study, the digital gene expression (DGE) profiles of the cultivated P. tenuifolia were analyzed in different phenological phase (flowering fruit bearing stage, wilting stage, dormancy stage). The differentially expressed genes were found in the biosynthesis of chemical composition in P. tenuifolia, and the representational ones were validated by RT-q PCR. Then, the key enzymes(CYP450s and UGTs) involved in the downstream of the triterpenoid saponins biosynthesis pathway in P. tenuifolia were predicted through the correlation analysis of gene expression. The number of down-regulated genes was more than that of up-regulated in P. tenuifolia from flowering fruit bearing stage to dormancy stage. Six differentially expressed genes (HMGS, PMK, FPPS, SQS, SE, ß-AS) and five (PAL, C4 H, 4CL, CAD, peroxidase) were annotated to the triterpenoid saponins and phenylpropanoid biosynthesis pathway in P. tenuifolia, respectively. Compared to wilting and dormancy stages, the saponins, xanthones, and lignins were largely synthesized at the flowering fruit bearing stage of P. tenuifolia. Furthermore, UGT83A1, CYP716B1, CYP98A3, CYP86B1, and CYP94A1 may be the part of key enzymes in the downstream of the triterpenoid saponins biosynthesis pathway in P. tenuifolia. This study provides evidence to support the correctness of traditional harvest time of P. tenuifolia at the level of transcription, and lays the scientific foundation for gene cloning and functional verification of CYP450 s and UGTs in the downstream of the triterpenoid saponins biosynthesis pathway in P. tenuifolia in the future.

[Rationality of commodity criteria and traditional breeding of Polygala tenuifolia based on agronomic traits and determination of chemical components].

The agronomic traits (plant height, root diameter, root length, first lateral root height, lateral root amount, root weight) of 18 Polygala tenuifolia samples with different agronomic traits were analyzed, respectively. HPLC was used to analyze three main characteristic components including tenuifolin, polygalaxanthone Ⅲ, and 3,6'-disinapoyl sucrose. At last, the correlation between six agronomic traits and three main characteristic components were analyzed by scatter plot. We found no significant correlation between root diameter and three main characteristic components. There were no obvious correlations between tenuifolin and the remaining five agronomic traits. Short root length and first lateral root height as well as high lateral root amount resulted in high levels of polygalaxanthone Ⅲ in P. tenuifolia samples. High levels of 3,6'-disinapoyl sucrose were observed in P. tenuifolia samples with longer root. So, the current commodity criteria and traditional breeding of P. tenuifolia did not conform to pharmacopoeia standards, which excellent medicinal materials should have high contents of the main characteristic components. It was urgent to revise the current commodity criteria and breeding methods.

[UPLC/Q-TOF MS-Based Metabolomics Analysis of Chemical uiterences in Different Polygala tenuifolia Varieties].

OBJECTIVE: The chemical differences of Polygala tenuifolia varieties-JinYuan 1 (JY1), FenYuan 2 (FY2) and traditional FenYang (FY) were studied, in order to provide reference for the breeding of Polygala tenuifolia. METHODS: The samples of JY1, FY2 and FY were subjected to ultra-high performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (Q-TOF MS) analysis. The obtained data were analyzed using Principal Component Analysis (PCA) and other statistical analysis methods, and differential metabolites were further figured out. RESULTS: Compared with FY,sucrose esters (such as sibiricoses A5 and tenuifoliside B) and oligosaccharides (such as tenuifoliose K) in JY1 and FY2 contributed more to the separation of Polygala tenuifolia varieties in the PCA score plot. Compared with JYl, The sugar esters (such as tenuifoliside B and tenuifoliside A) and oligosaccharides( such as tenuifoliose A) in the FY2 also contributed more to the separation of Polygala tenuifolia varieties in the PCA score plot. In addition, the relative contents of sibiricaxanthone A,3,6'-disinapoly sucrose and senegin III showed significant differences among FY, JY1 and FY2. CONCLUSION: As new Polygala tenuifolia varieties, JY1 and FY2 had certain differences and respective advantages on the chemical composition compared with FY,which could provide data support for the directional breeding of Polygala tenuifolia based on the contents of some active compounds.

Induction of apoptosis in hormone-resistant human prostate cancer PC3 cells by inactivated Sendai virus.

OBJECTIVE: Inactivated Sendai virus particle [hemagglutinating virus of Japan envelope (HVJ-E)] has a potential oncolytic effect due to its ability to induce apoptosis in tumor cells. However, the molecular mechanism of apoptosis induction in cancer cells mediated by HVJ-E has not been fully elucidated. This paper aims to investigate the underlying mechanism of apoptosis induction by HVJ-E in prostate cancer cells (PC3). METHODS: PC3 cells were treated with HVJ-E at various MOI, and then interferon-ß (IFN-ß) production, and the cell viability and apoptosis were detected by ELISA, MTT-based assay and flow cytometry, respectively. Next, the roles of Jak-Stat, MAPK and Akt pathways played in HVJ-E-induced apoptosis in PC3 cells were analyzed by immunoblot assay. To further evaluate the cytotoxic effect of HVJ-E on PC3 cells, HVJ-E was intratumorally injected into prostate cancers on BALB/c-nude mice, and the tumor volume was monitored for 36 days. RESULTS: HVJ-E induced IFN-ß production and activated Jak-Stat signaling pathway, which resulted in the activation of caspase-8, caspase-3, and PARP in PC3 prostate cancer cells post HVJ-E treatment. Furthermore, we observed for the first time that p38 and Jnk MAPKs in PC3 cells contributed to HVJ-E-induced apoptosis. In addition, intratumoral HVJ-E treatment displayed a direct inhibitory effect in an in vivo BALB/c nude mouse prostate cancer model. CONCLUSION: Our findings have provided novel insights into the underlying mechanisms by which HVJ-E induces apoptosis in tumor cells.

A Broad-Spectrum Antiviral Molecule, Protoporphyrin IX, Acts as a Moderator of HIV-1 Capsid Assembly by Targeting the Capsid Hexamer.

The capsid protein (CA), an essential component of human immunodeficiency virus type 1 (HIV-1), represents an appealing target for antivirals. Small molecules targeting the CAI-binding cavity in the C-terminal domain of HIV-1 CA (CA CTD) confer potent antiviral activities. In this study, we report that a small molecule, protoporphyrin IX (PPIX), targets the HIV-1 CA by binding to this pocket. PPIX was identified via in vitro drug screening, using a homogeneous and time-resolved fluorescence-based assay. CA multimerization and a biolayer interferometry (BLI) assay showed that PPIX promoted CA multimerization and bound directly to CA. The binding model of PPIX to CA CTD revealed that PPIX forms hydrogen bonds with the L211and E212 residues in the CA CTD. Moreover, the BLI assay demonstrated that this compound preferentially binds to the CA hexamer versus the monomer. The superposition of the CAI CTD-PPIX complex and the hexameric CA structure suggests that PPIX binds to the interface formed by the NTD and the CTD between adjacent protomers in the CA hexamer via the T72 and E212 residues, serving as a glue to enhance the multimerization of CA. Taken together, our studies demonstrate that PPIX, a hexamer-targeted CA assembly enhancer, should be a new chemical probe for the discovery of modulators of the HIV-1 capsid assembly. IMPORTANCE CA and its assembled viral core play essential roles in distinct steps during HIV-1 replication, including reverse transcription, integration, nuclear entry, virus assembly, and maturation through CA-CA or CA-host factor interactions. These functions of CA are fundamental for HIV-1 pathogenesis, making it an appealing target for antiviral therapy. In the present study, we identified protoporphyrin IX (PPIX) as a candidate CA modulator that can promote CA assembly and prefers binding the CA hexamer versus the monomer. PPIX, like a glue, bound at the interfaces between CA subunits to accelerate CA multimerization. Therefore, PPIX could be used as a new lead for a CA modulator, and it holds potential research applications.

Modulation of HIV-1 capsid multimerization by sennoside A and sennoside B via interaction with the NTD/CTD interface in capsid hexamer.

Small molecules that bind to the pocket targeted by a peptide, termed capsid assembly inhibitor (CAI), have shown antiviral effects with unique mechanisms of action. We report the discovery of two natural compounds, sennoside A (SA) and sennoside B (SB), derived from medicinal plants that bind to this pocket in the C-terminal domain of capsid (CA CTD). Both SA and SB were identified via a drug-screening campaign that utilized a time-resolved fluorescence resonance energy transfer assay. They inhibited the HIV-1 CA CTD/CAI interaction at sub-micromolar concentrations of 0.18 µM and 0.08 µM, respectively. Mutation of key residues (including Tyr 169, Leu 211, Asn 183, and Glu 187) in the CA CTD decreased their binding affinity to the CA monomer, from 1.35-fold to 4.17-fold. Furthermore, both compounds induced CA assembly in vitro and bound directly to the CA hexamer, suggesting that they interact with CA beyond the CA CTD. Molecular docking showed that both compounds were bound to the N-terminal domain (NTD)/CTD interface between adjacent protomers within the CA hexamer. SA established a hydrogen-bonding network with residues N57, V59, Q63, K70, and N74 of CA1-NTD and Q179 of CA2-CTD. SB formed hydrogen bonds with the N53, N70, and N74 residues of CA1-NTD, and the A177and Q179 residues of CA2-CTD. Both compounds, acting as glue, can bring αH4 in the NTD and αH9 in the CTD of the NTD/CTD interface close to each other. Collectively, our research indicates that SA and SB, which enhance CA assembly, could serve as novel chemical tools to identify agents that modulate HIV-1 CA assembly. These natural compounds may potentially lead to the development of new antiviral therapies with unique mechanisms of action.

Natural-product-library-based screening for discovery of capsid C-terminal domain targeted HIV-1 inhibitors.

Antiretroviral therapy (ART) can effectively suppress replication of human immunodeficiency virus type 1 (HIV-1) and limit disease progression. However, ART is unable to eradicate the virus, and the requirement for lifelong treatment may have side effects and may lead to the development of resistance. New approaches to prevent and treat HIV-1 infection should therefore be developed. HIV-1 capsid (CA) protein is an unexploited but attractive target for antiviral drug development. The hydrophobic cavity of the C-terminal domain of CA (CA CTD) has been validated as a potential target for antiviral drugs. Binding of compounds to this conserved non-polar groove in CA CTD allosterically disrupts the CA assembly. This study screened 2080 natural products to identify potential antiviral agents for further development to combat HIV-1 infection. From the primary screen at a fixed concentration of 50 µM, 16 compounds were found to be effective against this target. Six compounds observed in the primary screen were confirmed in dose-response experiments, and were tested against HIV-1-induced cytopathic effects. Two compounds were found to inhibit HIV-1 replication, and the most active compound - rubranol - inhibited viral replication at a moderate micromolar concentration (EC50 = 15.85 µM). The binding modes of rubranol and hirsutanonol to CA CTD were analysed by molecular docking, providing insight for the design of drugs targeting HIV-1 CA. This study reports, for the first time, identification of natural products that showed potential as anti-HIV-1 agents by targeting the conserved hydrophobic cavity of HIV-1 CA CTD.

A HTRF based competitive binding assay for screening specific inhibitors of HIV-1 capsid assembly targeting the C-Terminal domain of capsid.

Due to its multifaceted essential roles in virus replication and extreme genetic fragility, the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein is a valued therapeutic target. However, CA is as yet unexploited clinically, as there are no antiviral agents that target it currently on the market. To facilitate the identification of potential HIV-1 CA inhibitors, we established a homogeneous time-resolved fluorescence (HTRF) assay to screen for small molecules that target a biologically active and specific binding pocket in the C-terminal domain of HIV-1 CA (CA CTD). The assay, which is based on competition of small molecules for the binding of a known CA inhibitor (CAI) to the CA CTD, exhibited a signal-to-background ratio (S/B) > 10 and a Z' value > 0.9. In a pilot screen of three kinase inhibitor libraries containing 464 compounds, we identified one compound, TX-1918, as a low micromolecular inhibitor of the HIV-1 CA CTD-CAI interaction (IC50 = 3.81 µM) that also inhibited viral replication at moderate micromolar concentration (EC50 = 15.16 µM) and inhibited CA assembly in vitro. Based on the structure of TX-1918, an additional compound with an antiviral EC50 of 6.57 µM and cellular cytotoxicity CC50 of 102.55 µM was obtained from a compound similarity search. Thus, the HTRF-based assay has properties that are suitable for screening large compound libraries to identify novel anti-HIV-1 inhibitors targeting the CA CTD.