DCF, a molecular weapon against bacterial defenses

Scientific discoveries are like great stories: they often begin with an encounter. Nearly 20 years ago, Professor Stéphane Vincent of UNamur's Laboratoire de Chimie Bio-Organique, then a young sugar chemist, was in search of something new. During a post-doctorate in Strasbourg, France, in the laboratory of Jean-Marie Lehn, winner of the 1987 Nobel Prize in Chemistry and a specialist in supramolecular chemistry, he befriended another post-doctoral fellow: the Romanian Mihail Barboiu, now a CNRS researcher in Montpellier.

Soon before the Covid-19 pandemic, at a scientific congress, Mihail Barboiu showed Stéphane Vincent the results of his experiments. "He was using DCFs as a kind of transporter, to bring genes (DNA or RNA fragments) into a cell", recalls the chemist. "I then realized that DCFs were positively-charged molecules and readily adapted to DNA, which is negatively-charged. This gave me the idea of using them against bacteria, in the same way as certain antibiotics, which are also positively charged."

The two researchers then established an initial research project, with a thesis funded in cotutelle by UNamur, which culminated in 2021 in the publication of the first results showing the antibacterial activity of DCFs. "At the time, I was already working on antibacterial approaches, particularly against Pseudomonas aeruginosa, a major pathogen that forms biofilms", explains Stéphane Vincent.

To combat antiseptics and antibiotics, bacteria proceed in several ways. In addition to developing mechanisms to block the functioning of antibiotics, they are able to aggregate or dock themselves to a surface, for example that of a medical implant, and cover themselves with a complex tangle of all sorts of molecules. The latter, known as biofilm, protects the bacteria from external aggression. These biofilms are a major public health problem, as they enable bacteria to survive even the most powerful antibiotics and are notably the cause of nosocomial diseases, infections contracted during a stay in a healthcare establishment.

"We have shown that certain DCFs are both capable of inhibiting biofilm production, but also of weakening them, thereby exposing bacteria to their environment", summarizes Stéphane Vincent.

Bolstered by these results and thanks to C2W, a "very competitive"European program that funds post-doctorates, Stéphane Vincent invited Dmytro Strilets, a Ukrainian chemist who had just completed his thesis under the supervision of Mihail Barboiu, to work in his laboratory on DCFs. The project, called TADAM and carried out in collaboration with researchers Tom Coenye of UGent and Charles Van der Henst of the VUB, then focused on the antibacterial and antibiofilm potential of DCFs against Acinetobacter baumannii, a bacterium which, along with Pseudomonas aeruginosa, is on the list of pathogens of greatest concern defined by the World Health Organization (WHO).

The TADAM project is based on an ingenious assembly: DCFs are associated with special molecules known as pillarenes. The latter form a sort of cage around a proven antibiotic molecule, levofloxacin, thus improving its bioavailability and stability. The DCFs then have the role of inhibiting and disintegrating the biofilm, to enable the pillarenes to deliver their antibiotic directly to the bacteria thus exposed.

The results obtained by Stéphane Vincent's team are spectacular: the DCF-pillararene-antibiotic assembly is up to four times more effective than the antibiotic used alone! Noting that little work had yet been done on the antibiotic effect of these new molecules, the researchers decided to protect their invention by filing a joint patent, before going any further.

For everything still remains to be done. Firstly, because despite more than convincing results, how the assembly works is still obscure. "All the study of the mechanism of action has yet to be done, says Stéphane Vincent. "How is the antibiotic arranged in the pillararene cage? Why do DCFs have antibiofilm activity? How do DCFs and pillararenes fit together? All these questions are important, not only to understand our results, but also to eventually develop new generations of molecules."

And on this point, Stéphane Vincent wants to be particularly cautious. "We all dream, of course, of a universal molecule that will work on all pathogens, but we have to be humble, he pauses. "I've been working with biologists for many years, and I know that biological reality is infinitely more complex than our laboratory conditions. But it's because our results are so encouraging that we must persevere down this path."

The chemist already has several leads: "We're going to test the molecules on bacteria"circulating"suspended in a liquid, which behave very differently. And then we're also going to work on clinical isolates of pathogenic bacteria, to get a little closer to the real conditions under which these biofilms form."

Dmytro Strilets has just received a Chargé de Recherche mandate from the FNRS to develop second-generation DCFs and study their mode of action. The TADAM project has received funding from the University of Namur and the European Union's Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement n°101034383.