Leena Pohjala

Inhibitors of alphavirus entry and replication identified with a stable Chikungunya replicon cell line and virus-based assays

Chikungunya virus (CHIKV), an alphavirus, has recently caused epidemic outbreaks and is therefore considered a re-emerging pathogen for which no effective treatment is available. In this study, a CHIKV replicon containing the virus replicase proteins together with puromycin acetyltransferase, EGFP and Renilla luciferase marker genes was constructed. The replicon was transfected into BHK cells to yield a stable cell line. A non-cytopathic phenotype was achieved by a Pro718 to Gly substitution and a five amino acid insertion within non-structural protein 2 (nsP2), obtained through selection for stable growth. Characterization of the replicon cell line by Northern blotting analysis revealed reduced levels of viral RNA synthesis. The CHIKV replicon cell line was validated for antiviral screening in 96-well format and used for a focused screen of 356 compounds (natural compounds and clinically approved drugs). The 5,7-dihydroxyflavones apigenin, chrysin, naringenin and silybin were found to suppress activities of EGFP and Rluc marker genes expressed by the CHIKV replicon. In a concomitant screen against Semliki Forest virus (SFV), their anti-alphaviral activity was confirmed and several additional inhibitors of SFV with IC50 values between 0.4 and 24 µM were identified. Chlorpromazine and five other compounds with a 10H-phenothiazinyl structure were shown to inhibit SFV entry using a novel entry assay based on a temperature-sensitive SFV mutant. These compounds also reduced SFV and Sindbis virus-induced cytopathic effect and inhibited SFV virion production in virus yield experiments. Finally, antiviral effects of selected compounds were confirmed using infectious CHIKV. In summary, the presented approach for discovering alphaviral inhibitors enabled us to identify potential lead structures for the development of alphavirus entry and replication phase inhibitors as well as demonstrated the usefulness of CHIKV replicon and SFV as biosafe surrogate models for anti-CHIKV screening.

Aggregating Behavior of Phenolic Compounds - A Source of False Bioassay Results?

Previous descriptions of quercetin, a widely studied flavonoid, as a frequently reported nonspecific screening hit due to aggregating behavior has raised questions about the reliability of in vitro bioactivity reports of phenolic compounds. Here a systematic study on 117 phenolic compounds is presented, concerning their aggregating tendency and the relevance of this phenomenon to obtaining false bioassay results. Fourteen compounds formed aggregates detectable by dynamic light scattering (DLS) when assayed at 10 µM in Tris-HCl pH 7.5. Flavonoids were more prone to aggregation than other phenolic compounds, and the aggregate formation was highly dependent on the vehicle, ionic strength and pH. The compounds were also assayed against three unrelated enzymes in the presence and absence of Triton X-100, and their bioactivity ratios were collected from PubChem database. By comparing these datasets, quercetin and rhamnetin were confirmed as promiscuous inhibitors. In general, flavonoids exhibited also higher bioactivity ratios in the PubChem database than coumarins or organic acids. To conclude, aggregate formation can be controlled with Triton X-100 and this phenomenon needs to be considered when bioassay data is interpreted, but our data indicates that it does not always lead to unspecific inhibition of biological targets.

Inhibitory effect of the natural product betulin and its derivatives against the intracellular bacterium Chlamydia pneumoniae.

Chlamydia pneumoniae is a universal pathogen that has been indicated to play a part in the development of asthma, atherosclerosis and lung cancer. The complete eradication of this intracellular bacterium is in practice impossible with the antibiotics that are currently in use and studies on new antichlamydial compounds is challenging because Chlamydia research lacks the tools required for the genetic modification of this bacterium. Betulin is a natural lupane-class triterpene derived from plants with a wide variety of biological activities. This compound group thus has wide medical potentials, and in fact has been shown to be active against intracellular pathogens. For this reason, betulin and its derivatives were selected to be assayed against C. pneumoniae in the present study. Thirty-two betulin derivatives were assayed against C. pneumoniae using an acute infection model in vitro. Five promising compounds with potential lead compound characteristics were identified. Compound 24 (betulin dioxime) gave a minimal inhibitory concentration (MIC) of 1 microM against strain CWL-029 and showed activity in nanomolar concentrations, as 50% inhibition was achieved at 290 nM. The antichlamydial effect of 24 was confirmed with a clinical isolate CV-6, showing a MIC of 2.2 microM. Previous research on betulin and its derivatives has not identified such a remarkable inhibition of Gram-negative bacterial growth. Furthermore, we also demonstrated that this antichlamydial activity was not due to PLA(2) (EC 3.1.1.4) inhibition caused by the betulin derivatives.

Effects of coadministration of natural polyphenols with doxycycline or calcium modulators on acute Chlamydia pneumoniae infection in vitro

Besides small molecules from medicinal chemistry, natural products are still major sources of innovative therapeutic agents for various conditions, including infectious diseases. Here we present the first attempt to design a combination treatment targeted against Chlamydia pneumoniae infection using coadministration of natural phenolics with calcium (Ca2+) modulators, and also the concomitant administration of these compounds with doxycycline. An in vitro acute C. pneumoniae model in human lung epithelial cells was used and Loewe additivity model was applied to evaluate the effects. In general, the phenolic compounds, quercetin, luteolin, rhamnetin and octyl gallate did not improve the antichlamydial effect of doxycycline, and, in some cases, resulted in antagonistic effects. The combination of doxycycline and Ca2+ modulators (isradipine, verapamil and thapsigargin) was at most additive, and at subinhibitory concentrations of doxycycline, often even antagonistic. The Ca2+ modulators showed no inhibitory effects on C. pneumoniae growth alone, whereas the coadminstration of Ca2+ modulators with phenolic compounds resulted in potentiation of the antichlamydial effect of phenolic compounds. Verapamil (100 μM) was synergistic with low quercetin and luteolin concentrations (0.39 and 1.56 μM), whereas 10 μM isradipine was synergistic with high quercetin, rhamnetin and octyl gallate concentrations (12.5 μM and 100 μM). Use of thapsigargin with the phenolic compounds resulted in the most intense synergism. Interaction indices 0.12 and 0.14 were achieved with 0.39 μM luteolin and 10 and 100 nM thapsigargin, respectively. To conclude, the observed results indicate that the Ca2+ modulators potentiate the antichlamydial effects of the phenolic compounds.

Comparison of transgenic Gerbera hybrida lines and traditional varieties shows no differences in cytotoxicity or metabolic fingerprints

Genetic modification using gene transfer (GM) is still controversial when applied to plant breeding at least in Europe. One major concern is how GM affects other genes and thus the metabolism of the plant. In this study, 225 genetically modified lines of the ornamental plant Gerbera hybrida and 42 non-GM gerbera varieties were used to investigate changes in secondary metabolism. The cytotoxicity of GM and non-GM gerbera extracts was evaluated on human cell lines derived from lung, liver, and intestinal tissues. The results indicate that the safety profile for GM gerbera lines is similar to the viability pattern for non-GM varieties—none of the extracts were toxic. In addition, metabolic fingerprints of gerbera extracts were identified using thin-layer chromatography and analysed by principal component analysis (PCA), the nearest neighbour classifier, and Fligner-Killeen test. No new compounds unique to GM lines were observed. With PCA, no separation between GM gerbera lines and varieties could be demonstrated. In the nearest neighbour classifier, 54% of the samples found the expected neighbour based on the gene constructs used for transformation. With Fligner-Killeen test, we studied if the amounts of compounds vary more in GM gerberas than in varieties. In most cases, there were no statistically significant differences between the varieties and GM lines or there was more variation among the non-GM varieties than in the GM lines. The variance of a single compound was significantly larger in transgenic gerbera lines than in varieties and of three compounds in non-GM varieties.