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.
