Extracting new activities from complexity
The search for new active compounds in a vast collection of diverse microorganisms starts with a well designed screening process. The activity testing unit takes on the challenge of systematically screening hundreds of bacterial extracts from DEINOVE’s collection of strains for new compounds with antimicrobial activity.
The screening process combines high throughput and automated technologies to assay crude extracts in a battery of biological assays. Interesting extracts that score positive during screening then undergo dereplication, an iterative process that involves several units (bioprocess engineering, advanced analytics, synthetic biology, data science and fermentation engineering) to ultimately identify the chemical entity that is responsible for an observed activity. While the activity testing hub mainly concentrates in the initial screening, it also plays an important role throughout the development process to:
- Confirm the presence of an activity,
- Identify the most promising compounds to move into preclinical studies,
- Evaluate the mechanisms of action of the identified molecules,
- Characterize the potential mechanisms of resistance to the compound.
Screening for new antimicrobial activities
Bacterial strains or extracts provided by the biodiversity farming unit are tested for an activity against various pathogens, such as gram positive and gram negative bacteria and fungi. For example, DEINOVE focuses on the “ESKAPE” pathogens commonly found to carry antimicrobial resistance genes: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species.
Several methods are used in the screening process, at different stages, to evaluate the strains or extracts such as:
- Automated thin-layer chromatography (TLC) coupled to bioautography. The chromatography separates compounds within a crude bacterial extract by migration on silica plates, while the bioautography reveals antimicrobial activities against different pathogens across the migration lanes. Because this qualitative technique is separative, it can detect several active molecules in an extract, as well as provide information that will be valuable towards their identification (size, physico-chemical properties…).
- Automated antimicrobial susceptibility assay to assess minimal inhibitory concentrations (MIC). Bacterial extracts are serially diluted in microtiter plates and incubated with known pathogens to determine the lowest concentration that prevents visible growth of the pathogen. This assay is quantitative, highly reproducible, and can be scaled up for high-troughput screening of extracts on an extended panel of microorganisms.
- Microfluidics-based assays, such as antimicrobial susceptibility assays
- Eukaryotics cell based assays, such as cytotoxicity assays
Overall, the activity testing hub assigns a score to strains with antimicrobial activity on the basis of several criteria and determines which extracts should move on to dereplication.
- Customized development of assays that will be required for bioactivity-guided dereplication.
- Quality control of a biological activity during the compound development process.
- In parallel to preclinical development of a lead compound, the activity testing unit performs various assays to predict the emergence of resistance to the compound: serial passage assays, frequency of resistance as well as cross-resistance.
- To shed light on the mechanism of action of an antimicrobial compound and support its preclinical development, the activity testing hub also performs host-pathogen interaction studies.
Dewanjee, S., Gangopadhyay, M., Bhattacharya, N., Khanra, R., & Dua, T. K. (2015). Bioautography and its scope in the field of natural product chemistry. Journal of Pharmaceutical Analysis, 5(2), 75–84.
Hubert, J., Nuzillard, J.-M., & Renault, J.-H. (2015). Dereplication strategies in natural product research: How many tools and methodologies behind the same concept? Phytochemistry Reviews, 16(1), 55–95.