University of Helsinki, Helsinki 2006
A study on bacteria-targeted screening and in vitro safety assessment of natural products
Doctoral dissertation, April 2006.
More and more drugs are becoming useless as a result of increasing numbers of drug resistant pathogen bacteria strains. The urge to find new active drugs, pure or modified, has become a critical task to overcome the limitations that older, still in use, drugs have faced. Drug companies and research facilities are screening different sources with different techniques to fill this need. For a successful screening process, optimized high-quality methods are needed.
In this study, erythromycin resistant Streptococcus pyogenes strains, mefA, ermB and ermTR, and the Staphylococcus simulans ermC strain of Finnish origin were used to optimize a turbidometric screening assay using a 96-well microplate for detecting new antimicrobials. Optimization was assisted by using quality parameters S/N (signal-to-noise), S/B (signal-to-background) and Z' factor (screening window coefficient) to confirm the reliability and repeatability. The optimized assay was used for screening a small-scale library of natural compounds and their derivatives against the antibiotic resistant strains. The results showed that gallic esters, specially octyl gallate, had potential inhibition effect against tested strains. Lichen acids were also found to be good inhibitors against all tested strains.
The search for novel antibacterial agents can be facilitated by virtual screening of compound databases against a known bacterial target. Following this approach, approx. 200 000 compounds were screened in silico for binding to ErmC' and used for selecting the 49 best-binding, drug-like compounds for in vitro evaluation. As a primary screen, a fluorometric, biochemical assay measuring the inhibition of catechol-O-methyltransferase (COMT), structurally very similar to ErmC', was employed to evaluate the potential activity against ErmC. Out of the selected 49 compounds, two structurally very similar compounds were identified as confirmed hits with reasonable activity (IC50 values of 26 and 73 ÁM). However, no marked activity was observed in a cell-based assay performed with the Staphylococcus aureus ermC strain.
High-performance liquid chromatography (HPLC) was used for microfractionation of natural extracts to overcome limitations of photometric measurement of colored samples that can affect the results of a screening assay. The microfractionation was successfully combined with the 96-well microplate antibacterial assay. The study demonstrated that the use of microfractionation coupled with bioactivity screening is a powerful tool for the identification of active components in natural extracts.
To study natural extracts and their safety, a miniaturized Ames test with Salmonella typhimurium TA98 and TA100 strains in a 6-well plate was used. With a miniaturized method, the cost of the test can be decreased, and less time, workspace and amounts of compound are needed than in a normal Ames test. The assay was used to screen mutagenicity and antimutagenicity of rapeseed, pine bark and raspberry extracts and their factions with vinylsyringol, a pure compound from crude rapeseed oil. None of the extracts were shown to be mutagenic. When the metabolic activator (rat liver S9 enzyme) was not added with known positive control (mutagen) and extract, all of the extracts were observed to have antimutagenic properties.
The natural extracts were further studied with the Caco-2 model to evaluate their ability to affect the permeability of co-administrated drugs across the cell monolayer. It has been previously shown that some natural extracts can have drug interactions and affect the drug's cellular permeability. Here, the permeability of verapamil, ketoprofen, metoprolol, and paracetamol under the influence of co-administered natural extracts were studied. As a result, none of the extracts had notable effects.
In conclusion, it is important to have a proper approach when screening natural products for biological activity. Using the latest technology can be the key for finding new promising drug candidates. Assay validation and miniaturization are good ways to get results quickly and with less money and work. High-quality methods and a thorough investigation that also take safety aspects into account can have a significant effect on the overall success of the screening process.
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© University of Helsinki 2006
Last updated 11.04.2006