AbstractsBiology & Animal Science

Cell-based bioluminescent high-throughput screening methods in antibacterial drug discovery

by Susanna Nybond

Institution: University of Helsinki
Department: Faculty of Pharmacy; Centre for Drug Research;Division of Pharmaceutical Biosciences
Year: 2015
Keywords: farmasi
Record ID: 1138393
Full text PDF: http://hdl.handle.net/10138/154752


Due to the emergence of multidrug resistant bacteria, bacterial infections are still a major healthcare problem. Many factors have led to a discovery void of new antibacterial agents, rendering the current antibiotic pipeline inadequate for future medical needs. For example, the outcomes from pure biochemical high-throughput screening (HTS) assays have, in many cases, not led to successful clinical compounds. Therefore cell-based assays might be a better choice for primary screening. However, the antibacterial cell-based assays in the current use often require long incubation times and they are not always amenable for miniaturization and automation for HTS. In this work, two screening assays based on recombinant bioluminescent E. coli strains were optimized and implemented in the screening of chemical libraries and natural products in antibacterial drug discovery. One of the recombinant bacterial strains was a strain which is sensitive towards transcriptional and translational inhibitors. The assay based on this strain was successfully miniaturized into 384-format using automatized liquid handling and was validated with a proof-of-concept library containing known drugs. This provided a means to perform a larger scale high throughput screen of a compound library. Based on the HTS hit structures, a ligand-based in silico screening of a virtual chemical library was employed for hit enrichment. The most active hits and the in silico selected compounds were further investigated in more detail. Natural products have been an important source in drug discovery, especially in the discovery of antibiotics in the current use. However, matrix effects such as colour or turbidity of natural product extracts can potentially cause interference in conventional absorbance based microbial growth inhibition assays. Also, conventional antibacterial assays are usually not sensitive enough for detecting very small concentrations in fractionated natural product extracts. The feasibility of bioreporter -based assays in antimicrobial screening of natural products was demonstrated by screening an in-house natural product library. One of the assays was also implemented for investigating the antibacterial properties of an extract from a fungal culture filtrate, which demonstrated the sensitivity of the assay for identification of active components from fractionated samples. In conclusion, sensitive and reproducible bioassays amenable for further miniaturization and automation were developed for antibacterial drug discovery. Compared to conventional antimicrobial testing, the bioreporter-based methods offer important improvements such as simultaneous data acquirement on antimicrobial activity, first indication of mode of action and significant reduction of assay time to 2-4 h compared to 24 h in standard susceptibility assays. The developed bioluminescent assays led to the improvement of compound throughput in antimicrobial screening: from hundreds of samples (natural product extracts and fractions) in manually performed assays in 96-well…