The technique makes it possible to evaluate the efficiency of new molecules in real time in bacteria
A tool based on luminescent energy transfer (BRET) was successfully tested for the first time in bacteria. The advantage of BRET is tracking where, how, and how efficiently potential new drugs bind to target proteins in real time in living bacteria. This process shortens the more conventional drug discovery steps and accelerates the search for new compounds with antibiotic action. A published research today (11) in the magazine ACS Infectious Disease was led by researchers from Center for Medicinal Chemistry (CQMED) in partnership with researchers from Canada. CQMED is supported by FAPESP.
To be effective, an antibiotic must overcome numerous obstacles in the bacteria. Starting with the external membranes that function as barriers. In addition, there are efflux pumps that force out antimicrobial agents and antibiotic-modifying enzymes, acting as defense mechanisms for bacteria. “For these reasons, finding compounds that circumvent all these barriers and that are also safe for the human host is far from trivial”, explains Rafael Couñago, main researcher at CQMED and author of the study.
The two main strategies for identifying and developing new antimicrobials are basically the biochemical assay and the cellular assay. In the first, the antimicrobial compound is tested only with the target protein purified from the bacteria to check for interaction. The second strategy is to apply the compound to the bacteria and check if it is capable of killing it.
Both approaches have weaknesses. The biochemical test does not guarantee that the compound will have the same behavior in the cell “The potency vitro of the compound is not always related to cellular activity”, explains Rebeka Fanti, author of the study and who developed the research at CQMED during her master's thesis in the Genetics and Molecular Biology Program at Unicamp.
The cellular assay does not make it clear what the mechanism of action was in the bacteria, which hinders the improvements of the compound. “It’s very difficult to know which target the compound is reaching,” explains Couñago.
The BRET technique (bioluminescence resonance energy transfer) is based on the exchange of energy in the form of bioluminescence. This technology was created in 1999 and, since then, has been used for various applications. “The interesting thing about this method is that we are able to evaluate the interaction of a target protein with a drug candidate in a live bacterial cell”, explains Fanti. The novelty opened a new horizon of studies in medicinal chemistry, allowing the study mainly in the so-called engagement assay, which evaluates the behavior of small molecules in the cell.
However, until then, the method had only been tested on mammalian cells grown in the laboratory. In this study, researchers tested on two human pathogens Escherichia coli, which can cause serious urinary tract infections, e Mycobacterium abscessus, responsible for infections in various tissues, including lungs and skin.
The technique consists of two steps. Firstly, through genetic engineering strategies, bacteria begin to produce a luminescence-emitting complex, formed by the junction of a target protein with a luciferase. This luciferase is originally found in abyssal shrimp and is capable of emitting blue light. Then, a light-receiving molecule, called a draw, is added to the culture medium containing bacteria. That draw enters the bacteria and binds directly to the target protein. It has the ability to absorb the blue light produced by luciferase and re-emit it in the form of red fluorescence. This exchange of energy in the form of light is called BRET. In this way, explains Fanti, small molecules or compounds can be evaluated and selected based on their ability to penetrate the bacteria and displace the draw of the target protein, causing a decrease in the BRET signal.
This study is particularly important for driving the development of new antibiotics and addressing the growing threat of antibiotic resistance among pathogenic bacteria. “The emergence of antimicrobial resistance far outstrips our current ability to discover, develop and approve new antimicrobials, especially those targeting Gram-negative pathogens and mycobacteria,” explains Couñago.
O Report Annual report by the World Health Organization (WHO) that analyzes the development stage of new antibacterial agents points out that the average time for a new antibiotic candidate to progress from the pre-clinical to the clinical stage is 10 to 15 years. Only one in 15 drug candidates will reach patients. For completely new antibiotics, this number is one in 30. Barriers to new product development include the long road to approval, high cost, and low success rates.
Method - To find out whether BRET would actually be effective for studying bacteria, the researchers carried out a proof of concept. To do this, they created a lineage of E.coli which produced the protein dihydrofolate reductase (DHFR), a target of antibiotics, added to a luciferase. The bacteria were then treated with an antibiotic of known efficiency, trimethoprim, which binds to DHFR. The result was the displacement of the draw by the antibiotic. In other words, the BRET method worked on bacteria. The researchers then tested a library of compounds on E.coli and even found a new compound with antibiotic potential.
Another important aspect for the action of an evaluated antibiotic is the time that the compound remains in the cell, that is, the retention time of the antibiotic. “It’s not enough to just enter, the molecule needs to accumulate in the bacterial cell to circumvent defense strategies”, explains Fanti. Using BRET, the researchers were also able to measure this parameter for trimethoprim. The test was also carried out on another type of bacteria, Mycobacterium abscessus, which has less permeable cell walls than those of E.coli. The results were equally positive.
Although all these results are promising, the technique has some limitations. Target protein ligands with known mechanisms of action must be available. “This information is crucial to developing a draw and add a luciferase to the target protein. Only then can the energy transfer, that is, BRET, happen”, points out Fanti. The next steps of the study are to expand the use of the technique to other bacterial pathogens and parasites.
ARTICLE: Rebeka C. Fanti, Stanley NS Vasconcelos, Carolina MC Catta-Preta, Jaryd R. Sullivan, Gustavo P. Riboldi, Caio V. dos Reis, Priscila Z. Ramos, Aled Edwards, Marcel A. Behr & Rafael M. Couñago. A Target Engagement Assay to Assess Uptake, Potency, and Retention of Antibiotics in Living Bacteria. ACS Infect. Dis. 2022. DOI: https://doi.org/10.1021/acsinfecdis.2c00073
