Mode of action of natural products in pathogenic Gram negative bacteria and their development as antibacterial agents

Main applicant Per Sunnerhagen
Co-applicants Anne Farewell, Jonas Warringer, Morten Grøtli, Edward Moore
Collaborator: Mate Erdelyi (Uppsala University)

Funding:
CARe (0,83 MSEK), VR research grant “Combatting antimicrobial resistance by novel antimalarial molecules against Plasmodium vivax and P. falciparum from South America and South-East Asia” and VR research grant “Exploration of East African Herbal Resources to Control Major Health Challenges”

Description
The global increase in antibiotics resistance calls for action on multiple fronts, one of which is to find new candidate antibiotics. Very few of these currently come out world-wide of the drug discovery pipeline. Trying to contribute to this effort, and potentially finding antibacterial molecules with new modes of action, we screen collections of molecules from tropical plants for antibacterial activity. Of particular interest are molecules active against Gram-negative bacteria.
The plants samples are collected in East Africa and South America in collaborative projects, and individual candidate molecules enriched and purified by chemical fractionation. We then screen all molecules for inhibition of growth of one Gram-negative and one Gram-positive species. Promising candidates are re-assayed to accurately quantitate their growth-inhibitory and cell-killing activity. The best molecules are then tested against a wider range of bacterial species mimicking more clinically relevant pathogens, and their respective activities determined. So far, we have identified five molecules with the most promising properties. Two of these are active against both Gram-negative and Gram-positive bacteria.
The next step will be to search for the mode of action of these molecules. Initially, we will use an unbiased method known as chemical genomics for this. Using a genome-wide collection of E. coli mutants, sensitivity profiles are obtained. From the patterns of sensitivity and resistance, the targeted function(s) in the bacterial cell can be inferred. If a particular molecule shows a cluster of affected mutants in one cellular function (e.g. DNA replication), then this is a strong indication that the mode of action involves that function. Based on these results, directed physiological and biochemical assays will be used to confirm the target protein/s.
For a new candidate antibacterial molecule, it will be important to determine if it is also active against bacteria that are resistant to existing antibiotics. We have on hand a wide range of bacterial strains isolated from different sources including clinical samples, which display different resistance patterns, and our candidate molecules will be tested against this strain battery. We will also look for potential synergies with established antibiotics. Further, any molecule to be taken forward in the process has to be tested for toxicity. So far, one molecule has been tested for cytotoxicity and shown to be relatively well tolerated. In the longer perspective, well-characterized and promising molecules may be chemically derivatized for increased antibacterial potency.