Periodic Reporting for period 1 - InECFAb (Inhibitors of ECF transporters as novel antibacterial agents)
Reporting period: 2024-04-01 to 2025-09-30
Antimicrobial resistance (AMR) is one of the most urgent global health threats. Resistant bacteria make the treatment of several infections difficult, leading to higher mortality and a significant economic burden. AMR could cause up to 10 million deaths per year worldwide by 2050, if no clear actions are taken.
One of the main causes for this crisis is the lack of new antibiotics with novel modes of action. To overcome this challenge, the InECFAb project focused on targeting the energy-coupling factor transporters (ECF-T) as a new antibacterial strategy. ECF-T are essential transmembrane proteins responsible for vitamin uptake in bacteria, but are absent in human cells, making them highly attractive drug targets.
The overall objective was to develop and optimize ECF-T inhibitors besides advanced target-engagement studies. The project also aimed to perform in vivo pharmacokinetics and early proof-of-concept studies, which could serve as a foundation for future novel antibacterial agents.
One of the main causes for this crisis is the lack of new antibiotics with novel modes of action. To overcome this challenge, the InECFAb project focused on targeting the energy-coupling factor transporters (ECF-T) as a new antibacterial strategy. ECF-T are essential transmembrane proteins responsible for vitamin uptake in bacteria, but are absent in human cells, making them highly attractive drug targets.
The overall objective was to develop and optimize ECF-T inhibitors besides advanced target-engagement studies. The project also aimed to perform in vivo pharmacokinetics and early proof-of-concept studies, which could serve as a foundation for future novel antibacterial agents.
The project built on promising hits identified during a previous ERC-funded work. In this project, we pursued a systematic multiparameter optimization where new derivatives were designed, synthesized, and tested in several iterative cycles. The derivatives demonstrated on-target activity together with enhanced antibacterial activity. The promising compounds did not show any in vitro cytotoxicity towards the HepG2 cell line up to 100 μM with high microsomal and plasma stability profiles.
Efforts were done with cryo-electron microscopy (EM) to obtain high-resolution structures of ECF-FolT2 with the inhibitors. Compounds with good potency and solubility were selected for structure determination. The resolution of the structures was at best 3.3 Å, which was probably insufficient to observe bound ligands. This also did not improve a lot with the use of Nanobodies. Finally, protocols for structure determination need to be improved to yield the required resolution to confidently identify bound ligands
We also demonstrated that the genes encoding ECF transporters are highly conserved in S. pneumoniae, and that their expression is crucial in bacterial infections. Furthermore, we showed that the antimicrobial activity of our inhibitors correlates with the ECF transporter expression by S. pneumoniae. Next, we conducted pharmacokinetic studies with the most promising derivatives, with candidates showing suitable bioavailability. One compound was evaluated in a neutropenic lung infection model against S. pneumoniae and led to a one log10-reduction in bacterial load compared to vehicle control.
Efforts were done with cryo-electron microscopy (EM) to obtain high-resolution structures of ECF-FolT2 with the inhibitors. Compounds with good potency and solubility were selected for structure determination. The resolution of the structures was at best 3.3 Å, which was probably insufficient to observe bound ligands. This also did not improve a lot with the use of Nanobodies. Finally, protocols for structure determination need to be improved to yield the required resolution to confidently identify bound ligands
We also demonstrated that the genes encoding ECF transporters are highly conserved in S. pneumoniae, and that their expression is crucial in bacterial infections. Furthermore, we showed that the antimicrobial activity of our inhibitors correlates with the ECF transporter expression by S. pneumoniae. Next, we conducted pharmacokinetic studies with the most promising derivatives, with candidates showing suitable bioavailability. One compound was evaluated in a neutropenic lung infection model against S. pneumoniae and led to a one log10-reduction in bacterial load compared to vehicle control.
ECF transporters are essential for bacterial survival and represent an unexplored mode of action for the development of antibacterial agents. In the project, the optimized derivatives showed potent antibacterial activity against Gram-positive bacteria, including drug-resistant pathogens. The results provide a new class of antibacterial agents, which, with further development, could address the AMR challenges.