PIK-24 Inhibits RSV-Induced Syncytium Formation via Direct Interaction with the p85α Subunit of PI3K.

Phosphoinositide-3 kinase (PI3K) signaling regulates many cellular processes, including cell survival, differentiation, proliferation, cytoskeleton reorganization, and apoptosis. The actin cytoskeleton regulated by PI3K signaling plays an important role in plasma membrane rearrangement. Currently, it is known that respiratory syncytial virus (RSV) infection requires PI3K signaling. However, the regulatory pattern or corresponding molecular mechanism of PI3K signaling on cell-to-cell fusion during syncytium formation remains unclear. This study synthesized a novel PI3K inhibitor PIK-24 designed with PI3K as a target and used it as a molecular probe to investigate the involvement of PI3K signaling in syncytium formation during RSV infection. The results of the antiviral mechanism revealed that syncytium formation required PI3K signaling to activate RHO family GTPases Cdc42, to upregulate the inactive form of cofilin, and to increase the amount of F-actin in cells, thereby causing actin cytoskeleton reorganization and membrane fusion between adjacent cells. PIK-24 treatment significantly abolished the generation of these events by blocking the activation of PI3K signaling. Moreover, PIK-24 had an obvious binding activity with the p85α regulatory subunit of PI3K. The anti-RSV effect similar to PIK-24 was obtained after knockdown of p85α in vitro or knockout of p85α in vivo, suggesting that PIK-24 inhibited RSV infection by targeting PI3K p85α. Most importantly, PIK-24 exerted a potent anti-RSV activity, and its antiviral effect was stronger than that of the classic PI3K inhibitor LY294002, PI-103, and broad-spectrum antiviral drug ribavirin. Thus, PIK-24 has the potential to be developed into a novel anti-RSV agent targeting cellular PI3K signaling. IMPORTANCE PI3K protein has many functions and regulates various cellular processes. As an important regulatory subunit of PI3K, p85α can regulate the activity of PI3K signaling. Therefore, it serves as the key target for virus infection. Indeed, p85α-regulated PI3K signaling facilitates various intracellular plasma membrane rearrangement events by modulating the actin cytoskeleton, which may be critical for RSV-induced syncytium formation. In this study, we show that a novel PI3K inhibitor inhibits RSV-induced PI3K signaling activation and actin cytoskeleton reorganization by targeting the p85α protein, thereby inhibiting syncytium formation and exerting a potent antiviral effect. Respiratory syncytial virus (RSV) is one of the most common respiratory pathogens, causing enormous morbidity, mortality, and economic burden. Currently, no effective antiviral drugs or vaccines exist for RSV infection. This study contributes to understanding the molecular mechanism by which PI3K signaling regulates syncytium formation and provides a leading compound for anti-RSV infection drug development.

Mechanism of Cross-Resistance to Fusion Inhibitors Conferred by the K394R Mutation in Respiratory Syncytial Virus Fusion Protein.

The fusion glycoprotein (F) is essential for respiratory syncytial virus (RSV) entry and has become an attractive target for anti-RSV drug development. Despite the promising prospect of RSV F inhibitors, issues of drug resistance remain challenging. In this study, we established a dual-luciferase protocol for RSV fusion inhibitor discovery. A small-molecule inhibitor, salvianolic acid R (LF-6), was identified to inhibit virus-cell and cell-cell fusion mediated by the RSV F protein. Sequence analysis of the resultant resistant viruses identified a K394R mutation in the viral F protein. The K394R mutant virus also conferred cross-resistance to multiple RSV fusion inhibitors, including several inhibitors undergoing clinical trials. Our study further showed that K394R mutation not only increased the triggering rate of F protein in prefusion conformation but also enhanced the fusion activity of F protein, both of which were positively correlated with resistance to fusion inhibitors. Moreover, the K394R mutation also showed cooperative effects with other escape mutations to increase the fusion activity of F protein. By substitution of K394 into different amino acids, we found that K394R or K394H substitution resulted in hyperfusiogenic F proteins, whereas F variants with other substitutions exhibited less fusion activity. Both K394R and K394H in F protein exhibited cross-resistance to RSV fusion inhibitors. Collectively, these findings reveal a positive correlation between the membrane fusion activity of F protein and the resistance of corresponding inhibitors. All of the results demonstrate that K394R in F protein confers cross-resistance to fusion inhibitors through destabilizing F protein and increasing its membrane fusion activity. IMPORTANCE Respiratory syncytial virus (RSV) causes serious respiratory tract disease in children and the elderly. Therapeutics against RSV infection are urgently needed. This study reports the discovery of a small-molecule inhibitor of RSV fusion glycoprotein by using a dual-luciferase protocol. The escape mutation (K394R) of this compound also confers cross-resistance to multiple RSV fusion inhibitors that have been reported previously, including two candidates currently in clinical development. The combination of K394R with other escape mutations can increase the resistance of F protein to these inhibitors through destabilizing F protein and enhancing the membrane fusion activity of F protein. By amino acid deletion or substitution, we found that a positively charged residue at the 394th site is crucial for the fusion ability of F protein, as well as for the cross-resistance against RSV fusion inhibitors. These results reveal the mechanism of cross-resistance conferred by the K394R mutation and the possible cross-resistance risk of RSV fusion inhibitors.

Inhibitory Effect of PIK-24 on Respiratory Syncytial Virus Entry by Blocking Phosphatidylinositol-3 Kinase Signaling.

Phosphoinositide-3 kinase signaling modulates many cellular processes, including cell survival, proliferation, differentiation, and apoptosis. Currently, it is known that the establishment of respiratory syncytial virus infection requires phosphoinositide-3 kinase signaling. However, the regulatory pattern of phosphoinositide-3 kinase signaling or its corresponding molecular mechanism during respiratory syncytial virus entry remains unclear. Here, the involvement of phosphoinositide-3 kinase signaling in respiratory syncytial virus entry was studied. PIK-24, a novel compound designed with phosphoinositide-3 kinase as a target, had potent anti-respiratory syncytial virus activity both in vitro and in vivo PIK-24 significantly reduced viral entry into the host cell through blocking the late stage of the fusion process. In a mouse model, PIK-24 effectively reduced the viral load and alleviated inflammation in lung tissue. Subsequent studies on the antiviral mechanism of PIK-24 revealed that viral entry was accompanied by phosphoinositide-3 kinase signaling activation, downstream RhoA and cofilin upregulation, and actin cytoskeleton rearrangement. PIK-24 treatment significantly reversed all these effects. The disruption of actin cytoskeleton dynamics or the modulation of phosphoinositide-3 kinase activity by knockdown also affected viral entry efficacy. Altogether, it is reasonable to conclude that the antiviral activity of PIK-24 depends on the phosphoinositide-3 kinase signaling and that the use of phosphoinositide-3 kinase signaling to regulate actin cytoskeleton rearrangement plays a key role in respiratory syncytial virus entry.

Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Antiviral drug development against respiratory syncytial virus (RSV) is urgently needed due to the public health significance of the viral infection. Here, we report the anti-RSV activity of a small molecule, (1S,3R,4R,5R)-3,4- bis{[(E)-3-(3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,5-dihydroxycyclohexane-1-carboxylic methyl ester (3,4-DCQAME) or 3,4- O-Dicaffeoylquinic acid methyl ester, which can be isolated from several plants of traditional Chinese medicine. We showed for the first time that compound 3,4-DCQAME potently inhibits RSV entry and infection. In vitro, 3,4-DCQAME can interact with F(ecto), the ectodomain of RSV fusion (F) protein. In cultured cells, the compound can block the interaction of F(ecto) protein with the cellular membrane and inhibit viral fusion during RSV entry, leading to inhibition of viral gene expression and infection. In RSV-infected mice that were treated with 3,4-DCQAME, we observed a reduction of RSV-induced pathologic changes and substantial inhibition of viral infection and growth in the lung tissues. Our results provide the first direct evidence of the anti-RSV activity of 3,4-DCQAME. Furthermore, these results suggest that 3,4-DCQAME represents a promising lead compound for anti-RSV therapeutic development.-Tang, W., Li, M., Liu, Y., Liang, N., Yang, Z., Zhao, Y., Wu, S., Lu, S., Li, Y., Liu, F. Small molecule inhibits respiratory syncytial virus entry and infection by blocking the interaction of the viral fusion protein with the cell membrane.

Macrocyclic Diterpenoids from Euphorbia helioscopia and Their Potential Anti-inflammatory Activity.

Guided by 1H NMR spectroscopic experiments using the aromatic protons as probes, 11 macrocyclic diterpenes (1-11) were isolated from the aerial parts of Euphorbia helioscopia. Their full three-dimensional structures, including absolute configurations, were established unambiguously by spectroscopic analysis and single-crystal X-ray crystallographic experiments. Among the isolated compounds, compound 1 is the third member thus far of a rare class of Euphorbia diterpenes featuring an unusual 5/10 fused ring system, and 2-4 are new jatrophane diterpenes. Based on the NMR data of the jatrophane diterpenes obtained in this study as well as those with crystallographic structures reported in the literature, the correlations of the chemical shifts of the relevant carbons and the configurations of C-2, C-13, and C-14 of their flexible macrocyclic ring were considered. Moreover, the anti-inflammatory activities of 1-11 were investigated by monitoring their inhibitory effects on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells. Compound 1 showed an IC50 of 7.4 ± 0.6 µM, which might be related to the regulation of the NF-κB signaling pathway by suppressing the translocation of the p65 subunit and the consequent reduction of IL-6 and TNF-α secretions.

Stilbene Glycoside Oligomers from the Roots of Polygonum multiflorum.

Five new trans-2,3,5,4'-tetrahydroxystilbene 2-O-ß-d-glucopyranoside (TSG)-based stilbene glycoside oligomers (1-5) were isolated from the roots of Polygonum multiflorum. Their structures were elucidated by comprehensive spectroscopic analyses and chemical evidences. The absolute configurations of 1, 2, 4, and 5 were established by quantum-chemical electronic circular dichroism (ECD) calculations. Putative biosynthetic pathways of 1-5 were proposed using TSG as the key precursor. In addition, compounds 1 (multiflorumiside H) and 3 (multiflorumiside J) exhibited moderate inhibitory activities against NO production in LPS-stimulated RAW264.7 cells.

Eleven New Triterpenoid Glycosides from the Roots of Ilex asprella.

Asprellosides A-K, nine new ursane-type triterpenoid glycosides (1-9), and two new oleanane-type triterpenoid glycosides (10 and 11), including six rare sulfated triterpenoid glycosides, were isolated from the roots of Ilex asprella. Their structures were determined on the basis of comprehensive spectroscopic analysis and chemical methods. Among these compounds, asprelloside B (2) and asprelloside C (3) are the first examples of triterpenoid glycosides bearing a rare 3,4-O-disulfo-xylopyranosyl residue. All the saponins isolated showed no significant effects against respiratory syncytial virus (RSV) and lipopolysaccharide-induced nitric oxide production in Raw264.7 macrophages.

Phloroglucinol Derivatives with Unusual Skeletons from Cleistocalyx operculatus and Their in Vitro Antiviral Activity.

Four novel phloroglucinol derivatives (1-4) featuring a 2,4-dimethyl-cinnamyl-phloroglucinol moiety, along with their putative biosynthetic precursors 5 and 6, were isolated from the leaves of Cleistocalyx operculatus. Compounds 1 and 2 are two pairs of new enantiomeric phloroglucinol dimers possessing an unprecedented polycyclic skeleton with a highly functionalized dihydropyrano[3,2- d]xanthene tetracyclic core. Compounds 3 and 4 are two new phloroglucinol-terpene adducts (PTAs) with a novel carbon skeleton. The structures of 1-4 including their absolute configurations were unambiguously accomplished by combination of extensive spectroscopic analyses, X-ray crystallography, and quantum chemical ECD calculations. A hypothetical biosynthetic pathway for 1-4 was also proposed. Compound 1 exhibited a promising in vitro antiherpes simplex virus type-1 (HSV-1) effect.

Multiflorumisides A-G, Dimeric Stilbene Glucosides with Rare Coupling Patterns from the Roots of Polygonum multiflorum.

Multiflorumisides A-G (1-7), seven new dimeric stilbene glucosides with two rare coupling patterns, were isolated from the roots of Polygonum multiflorum. The structures of these new dimeric stilbene glucosides were elucidated through comprehensive spectroscopic and chemical analyses. The absolute configurations of 3 and 5-7 were established by comparing their experimental and quantum-chemical ECD data. Putative biosynthetic pathways toward the dimers and their suppressive effects against nitric oxide production in lipopolysaccharide-stimulated RAW264.7 cells are also discussed.

Total Synthesis of the Flavonoid Natural Product Houttuynoid A.

The first total synthesis of the antiviral flavonoid houttuynoid A (1) has been achieved from aryl ketone 6 and benzofuran aldehyde 5 in nine linear steps. The C6-C3-C6 structure of the flavonoid was synthesized by an I2-catalyzed oxa-Michael addition of a chalcone intermediate, generated by the Claisen-Schmidt condensation of 5 and 6. This work provides a method for the synthesis of houttuynoids and provides a reference for the synthesis of the remaining members of the houttuynoid family.

Rearranged Phloroglucinol-Monoterpenoid Adducts from Callistemon rigidus.

Callisretones A (1) and B (2), two rearranged phloroglucinol-monoterpenoid adducts featuring an unprecedented isopropylcyclopenta[b]benzofuran backbone, together with their postulated biosynthetic precursors (3-9), were isolated from Callistemon rigidus. The previously assigned absolute configurations of viminalins H (7), L (8), and N (9) were revised and unequivocally established by X-ray diffraction data. A putative biosynthetic pathway toward callisretones A and B involving the rearrangement of the terpenoid motif is proposed. In addition, 1 and 2 showed inhibitory effects on nitric oxide production with IC50 values of 15.3 ± 1.0 and 17.7 ± 1.1 µM, respectively.

Matrine-Type Alkaloids from the Roots of Sophora flavescens and Their Antiviral Activities against the Hepatitis B Virus.

Eight new matrine-type alkaloids, flavesines G-J (1-4), alopecurine B (5), 7,11-dehydro-oxymatrine (6), 10-oxy-5,6-dehydromatrine (7), and 10-oxysophoridine (8), along with nine known analogues (9-17) were isolated from the roots of Sophora flavescens. Compounds 1-3 are the first natural matrine-type alkaloids with an open-loop ring D, while compound 4 represents an unprecedented dimerization pattern constructed from matrine and piperidine, and 5 is the first example of a matrine-type alkaloid with cleavage of the C-5-C-6 bond. The new structures were elucidated by means of spectroscopic data analysis (including NMR, MS, IR, and UV), and the absolute configurations were determined using single-crystal X-ray diffraction and ECD data. The isolated alkaloids were evaluated for their antiviral activity against hepatitis B virus, and compounds 1, 4, 5, 10, and 14 exhibited comparable antiviral potencies to matrine.

Antiviral Triketone-Phloroglucinol-Monoterpene Adducts from Callistemon rigidus.

Callistrilones F - K (1 - 6), six new triketone-phloroglucinol-monoterpene hybrids were isolated from the twigs and leaves of Callistemon rigidus. Their structures with absolute configurations were established by a combination analysis of NMR spectra, X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compounds 3 and 4 exhibited moderate inhibitory activities against herpes simplex virus (HSV-1) with IC50 values of 10.00 ± 2.50 and 12.50 ± 1.30 µm, respectively.

New Acetophenone Derivatives from Acronychia oligophlebia and Their Anti-inflammatory and Antioxidant Activities.

Seven new acetophenone derivatives (acroliones A - G, 1 - 7) and three known ones (8 - 10) were isolated from the leaves of Acronychia oligophlebia. Their structures were elucidated based on extensive spectroscopic analyses (IR, UV, HR-ESI-MS, 1D- and 2D-NMR), X-ray diffraction and comparison with literature data. The anti-inflammatory and antioxidant activities of all isolates were evaluated.

Anti-HSV Activity of Kuwanon X from Mulberry Leaves with Genes Expression Inhibitory and HSV-1 Induced NF-κB Deactivated Properties.

Six stilbene derivatives isolated from Mulberry leaves including Kuwanon X, Mulberrofuran C, Mulberrofuran G, Moracin C, Moracin M 3'-O-b-glucopyranoside and Moracin M were found to have antiviral effects against herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) at different potencies except for Mulberrofuran G. Kuwanon X exhibited the greatest activity against HSV-1 15577 and clinical strains and HSV-2 strain 333 with IC50 values of 2.2, 1.5 and 2.5 µg/mL, respectively. Further study revealed that Kuwanon X did not inactivate cell-free HSV-1 particles, but inhibited cellular adsorption and penetration of HSV-1 viral particles. Following viral penetration, Kuwanon X reduced the expression of HSV-1 IE and L genes, and decreased the synthesis of HSV-1 DNA. Furthermore, it was demonstrated that Kuwanon X inhibited the HSV-1-induced nuclear factor (NF)-κB activation through blocking the nuclear translocation and DNA binding of NF-κB. These results suggest that Kuwanon X exerts anti-HSV activity through multiple modes and could be a potential candidate for the therapy of HSV infection.

[Study on Anti-RSV Activities and QSAR of Natural Caffeoylquinic Acid Derivates].

Objective: To study the inhibitory effects of natural caffeoylquinic acid derivates against respiratory syncytial virus（ RSV） in vitro and their quantitative structure-activity relationship. Methods: The anti-RSV activities of caffeoylquinic acid derivates were detected by cytopathic effect（ CPE） method,MTT assay was adopted to evaluate the cytotoxicities of caffeoylquinic acid derivates and a three-dimensional quantitative structure-activity relationship（ 3D-QSAR） model was built. Results: The caffeoylquinic acid derivates showed different levels of anti-RSV activity. 4,5-d-CQAME（ CQA-4） was the most effective anti-RSV compound with IC50 of 2. 5µmol /L,and the SI was greater than 153. 8. Its effect was much better than the positive control drug ribavirin with the IC50 of 8. 3 µmol /L,and SI > 20,respectively. The result of 3D-QSAR study showed that caffeoyl group was important for the anti-RSV activity. Conclusion: Natural caffeoylquinic acid derivates exhibit anti-RSV activity and dicaffeoylquinic derivates are the most effective anti-RSV compounds. Both configuration and substituent groups have a great influence on the anti-RSV activity.

A new taraxastane-type triterpenoid glycoside from the roots of Clematis uncinata.

A new taraxastane-type triterpenoid glycoside, clematiunicinoside I (1), together with four known ones (2-5), was isolated from the roots of Clematis uncinata. The structure of the new compound was elucidated on the basis of spectroscopic analyses and acid hydrolysis. The cytotoxic activities of all the compounds against caski cervical cancer (Caski) cells were evaluated. This is the first report of the presence of taraxastane-type triterpenoid glycoside in the genus Clematis.

Triterpenoid saponins from the roots of Clematis uncinata.

Eight new bisdesmosidic triterpenoid saponins, clematiunicinosides A-H (1-8), along with eleven known ones (9-19), were isolated from the roots of Clematis uncinata. Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All the isolated saponins were tested for their cytotoxic activities on human caski cervical cancer (Caski) cells, and compounds 13, 17 and 19 exhibited inhibitory effect on Caski cells.

Triterpenoid saponins from the rhizomes of Anemone flaccida and their inhibitory activities on LPS-induced NO production in macrophage RAW264.7 cells.

A new ursane-type triterpenoid saponin, flaccidoside IV (1), and three new oleanane-type triterpenoid saponins, flaccidosides V-VII (2-4), along with 17 known saponins (5-21), were isolated from the rhizomes of Anemone flaccida. The structures of the new triterpenoid saponins were determined based on spectroscopic analyses and chemical methods. All the isolated saponins were tested for their inhibitory activities on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages, and several bisdesmosidic oleanane-type triterpenoid saponins (2, 7, and 10) showed significant inhibitory activities, which indicated they had potential anti-inflammatory activities under their noncytotoxic concentrations in vitro.

Chemical constituents from the roots and stems of Erycibe obtusifolia and their in vitro antiviral activity.

Three new quinic acid derivatives, 4-O-caffeoyl-3-O-sinapoylquinic acid methyl ester (1), 5-O-caffeoyl-4-O-syringoylquinic acid methyl ester (2), and 4-O-caffeoyl-3-O-syringoylquinic acid methyl ester (3), as well as four new coumarin glycosides, 7-O-(3-O-sinapoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (12), 7-O-(6-O-sinapoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (13), 7-O-(2-O-sinapoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (14), and 7-O-(6-O-syringoyl-ß-D-glucopyranosyl)-6-methoxycoumarin (15), together with eight known compounds (4-11) were isolated from the roots and stems of Erycibe obtusifolia. Their structures were elucidated on the basis of spectroscopic analysis and chemical evidence. All the compounds were screened for their in vitro antiviral activity against respiratory syncytial virus with a cytopathic effect reduction assay. Among them, the di-O-caffeoyl quinates 8-11 displayed a potent in vitro anti-respiratory syncytial virus effect.