The project focused on developing novel BamA‑inhibiting darobactin derivatives as a strategy to combat multidrug‑resistant Gram‑negative bacteria, particularly Pseudomonas aeruginosa. The first milestone established a comprehensive criteria catalogue that defined the requirements for a proprietary chemical scaffold and derived minimal and optimal target product profiles (TPPs). This scaffold was then used to generate a series of amino‑acid substitutions on the darobactin core. A freedom‑to‑operate (FtO) analysis, performed in collaboration with patent attorneys, confirmed that Darobactin B and its analogues were not covered by existing patents, thereby clearing the path for further development.
A detailed Gantt chart was produced to map the pre‑clinical development pathway and to quantify the financial needs for the next phase. This planning was instrumental in securing a follow‑up grant from the GoBio initial programme, which provided €1.2 million for 2023–2024. The grant enabled the retention and further training of the two Post‑doctoral researchers and funded consumables and travel to the GoBio workshop in Berlin.
The core experimental work involved creating a highly susceptible reporter strain that could detect BamA‑inhibiting activity. This strain was used to screen a library of biosynthetically produced darobactin derivatives. High‑throughput screening of a sequence library against the reporter strain identified the most potent amino‑acid combinations. Subsequent testing of these active molecules against a panel of over 50 clinical isolates of multidrug‑resistant P. aeruginosa revealed a MIC_80 of 8 µg mL^−1 for Darobactin B and its analogue Darobactin B9, demonstrating strong antibacterial activity within the defined TPP.
The project’s scientific output has been disseminated through several peer‑reviewed publications, including Cell Chemical Biology (2023), Angewandte Chemie (2023), Microbiology Spectrum (2023), and the Journal of the American Chemical Society (2022). These works describe the discovery of a dedicated halogenase involved in darobactin biosynthesis, the stabilization of a lateral‑closed BAM complex by Darobactin B, and the characterization of a radical SAM oxygenase responsible for ether cross‑linking in the molecule.
Collaboration was a cornerstone of the effort. The research team partnered with Myria Biosciences, led by Dr. Steven Schmitt, and the Pediatric Clinic of Dr. von Hauner Children’s Hospital at LMU Munich, headed by Dr. Ulrich von Both. These partners contributed expertise in clinical microbiology and translational research. Additionally, the project worked closely with Fraunhofer IME‑BR, which provided technical support and shared infrastructure. The GoBio initial programme, funded by the German government, served as the primary financial sponsor, while independent patent attorneys and scientific reviewers ensured the robustness of the FtO analysis and the novelty of the intellectual property.
Overall, the project achieved its technical objectives by establishing a robust platform for darobactin derivative discovery, demonstrating potent activity against clinically relevant pathogens, and securing substantial funding for continued pre‑clinical development. The collaborative framework, combining academic, industrial, and governmental stakeholders, positioned the project to advance toward the development of an innovative antibiotic capable of addressing the growing threat of multidrug‑resistant infections.