The work performed in WP 1 led to solving the crystal structures of most of the MEP pathway enzymes from several homologues. This includes the Mycobacterium tuberculosis (Mtb) DXPS, Plasmodium falciparum (Pf) DXPS, Pseudomonas aeruginosa (Pa) IspD, Klebsiella pneumoniae (Kp) IspD, PaIspE, Aquifex aeolicus (Aa) IspE, PaIspF, PaIspH, MtbIspH, and Escherichia coli (Ec) IspH. Additionally, several co-crystallization trials with inhibitors and substrates have been done resulting in complex structures of inhibitors with MtbDXR and PaDXPS while AaIspE was solved in complex with ATP. Optimum conditions for protein expression, purification and crystallization were established for several targets.
The drug-discovery process was initiated using different approaches: structure-, ligand- and fragment-based drug design; classical structure–activity relationship (SAR) studies and also more innovative named protein-templated techniques (i.e. dynamic combinatorial chemistry and kinetic target-guided synthesis). This use of a unique combination of various hit-identification strategies increased the chances of finding successful candidates in a short time. Therefore, each ESR in WP 2, built a structurally diverse collection of hits using a platform of various hit-identification strategies. Currently, we have promising inhibitors for each of the MEP pathway enzymes in hand that are undergoing multiparameter optimization en route to the clinic. Patent applications will be filed once the clear frontrunner for each target has emerged.
WP 3 is continuously monitoring the in vitro and whole-cell activities of the compounds synthesised by the ESR of WP2. In the case of ESR 10 and ESR 11 that work with Mycobacterium tuberculosis and Plasmodium falciparum, both have been trained to work with bacterial pathogens under BSL3 conditions. ESR 10 which is working with M. tuberculosis got the approval to work with human primary cells and, infection experiments with Mtb-infected primary human macrophages were successfully started to determine the activity against intracellular mycobacteria. In parallel to the in vitro testing, ESR 11 established the isopentenyl diphosphate (IDP) rescue assay in P. falciparum to prove the target engagement of the most promising MEP inhibitors.
Progress was also achieved in the proteomic approaches that will help target the identification of the most potent compounds.
The key results, including high-resolution structures, validated inhibitors, and novel chemical scaffolds, will provide a solid foundation for the development of clinical candidates targeting AMR pathogens. The intersectoral collaborations will ensure that the outcomes are transferable to both academic and industrial settings.
Several dissemination activities have been used to reach the scientific community including publications in high-impact journals and presentations at international conferences. In addition, MepAnti engaged in several outreach activities such as Famelab competition, newspaper articles and open days at universities, which helped to raise public awareness about AMR and the importance of anti-infective research.
